U.S. patent number 6,235,711 [Application Number 09/202,831] was granted by the patent office on 2001-05-22 for cell adhesion ihibiting compounds.
This patent grant is currently assigned to Zeneca Limited. Invention is credited to Anand Swaroop Dutta.
United States Patent |
6,235,711 |
Dutta |
May 22, 2001 |
**Please see images for:
( Certificate of Correction ) ** |
Cell adhesion ihibiting compounds
Abstract
##STR1## ##STR2## Cyclic peptide of formula (1) where Xaa.sub.1
is selected from L-amino acids selected from Phe, Lys and Arg,
D-amino acids selected from Phe and Met, the L- and D-amino acid
optionally substituted on its .alpha.-carbon or its .alpha.-amino
group with a C.sub.1-4 alkyl group; and Melle; Xaa.sub.2, Xaa.sub.3
et Xaa.sub.4 are respectively Leu, Asp and Val, optionally
substituted on their .alpha.-carbon or .alpha.-amino group with a
C.sub.1-4 alkyl group; X.sup.1 is selected from D-amino acids
selected from Ala, Phe, Arg, Lys, Trp, hArg(Et).sub.2,
Orn(CHMe.sub.2), Orn(Me.sub.2), Lys(CHMe.sub.2) and Arg(Pmc),
optionally substituted on their .alpha.-carbon or .alpha.-amino
group with a C.sub.1-4 alkyl group; Formula (II);
NH(CH.sub.2).sub.5 CO; and NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO,
where y is 1 or 2; X.sup.2 is selected from D-amino acids selected
from Ala, Arg, Lys, His, hArg(Et).sub.2, Orm(CHMe.sub.2), and
Om(Me.sub.2), optionally substituted on their .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
NH(CH.sub.2)SCH.sub.2 CO; and NH(CH.sub.2).sub.x CO, where x is 2
or 3; Xaa.sub.5 and Xaa.sub.6 are each independently a D-amino acid
selected from Ala and Arg, optionally substituted on its
.alpha.-carbon or .alpha.-mino group with a C.sub.1-4 alkyl group;
p is 0 or 1; and q is 0 or when p is 1, q is 0 or 1; or a salt
thereof. The cyclic peptides inhibit the interaction of vascular
cell adhesion molecule-1 and fibronectin with integrin very late
antigen 4 (.alpha.4.beta.61) and of mucosal addressin cell adhesion
molecule-1 (MAdCAM-1) with integrin .alpha.4.beta.7. They have
therapeutic applications such as in rheumatoid arthrids, multiple
sclerosis, astlna, psoriasis, inflammatory bowel disease and
insulin-dependent diabetes.
Inventors: |
Dutta; Anand Swaroop
(Macclesfield, GB) |
Assignee: |
Zeneca Limited (London,
GB)
|
Family
ID: |
10795723 |
Appl.
No.: |
09/202,831 |
Filed: |
December 21, 1998 |
Foreign Application Priority Data
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|
|
Jun 21, 1996 [GB] |
|
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9613112 |
|
Current U.S.
Class: |
514/9.3;
514/21.1; 530/328; 530/317; 530/329; 530/333; 514/863 |
Current CPC
Class: |
C07K
14/78 (20130101); A61P 19/02 (20180101); A61P
25/28 (20180101); A61P 17/06 (20180101); A61P
29/00 (20180101); A61P 11/06 (20180101); A61K
38/00 (20130101); Y10S 514/863 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/78 (20060101); A61K
38/00 (20060101); A61K 038/12 () |
Field of
Search: |
;514/11,16,17,863
;530/317,328,329,333 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0341915 |
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Nov 1989 |
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EP |
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0422938 |
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Apr 1991 |
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EP |
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WO 92/00995 |
|
Jan 1992 |
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WO |
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WO 94/02445 |
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Feb 1994 |
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WO |
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WO 94/15958 |
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Jul 1994 |
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WO |
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WO 95/15973 |
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Jun 1995 |
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WO |
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WO 95/14714 |
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Jun 1995 |
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WO |
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96/00581 |
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Jan 1996 |
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WO |
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WO 96/06108 |
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Feb 1996 |
|
WO |
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96/20216 |
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Jul 1996 |
|
WO |
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9620216 |
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Jul 1996 |
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WO |
|
Other References
Wayner: "Activation-dependent recognition by hematopoietic cells of
the LDV sequence in the V region of fibronectin", Journal of Cell
Biology, vol. 116, No. 2, 1992, pp. 489-497, cited in the
application, see the whole document. .
Kiso: "Synthesis of ANP fragments with hypertensive action",
Chemical Abstracts, vol. 110, No. 3, Jan. 16, 1989; abstract No.
24283k, p. 592, col. 1; see abstract & Pept.Chem.,-1987 pp.
512-516. .
B.Weinstein; "Chemistry and Biochemistry of Amino Acids, Peptides
and Proteins", 1983, See p. 338-p. 341. .
Aumailley et al., "Arg-Gly=Asp constrained within cyclic
pentapeptides--Strong and selective inhibitors of cell adhesion of
vitronectin and laminin fragment P1", Federation of European
Biochemical Societies, Oct., 1991, pp. 50-54. .
Lublin, "Susceptibility to experimental allergic encephalomyelitis
in animal models of autoimmunity", Neurology and Neurosurgery,
1992, vol. 5, pp. 182-187. .
Bowen et al., "Disease-Modifying Anti-Rheumatic Drugs: Strategies
for Screening", Pharmac. Ther. 1992, vol. 56, pp. 287-306. .
Nakajima et al., Role of Vascular Cell Adhesion Molecule 1/Very
Late Activation Antigen 4 and Intercellular Adhesion Molecule
1/Lymphocyte Function-associated Antigen 1 Interactions in
Antigen-induced Eosinophil and T Cell Recruitment into the Tissue.
J. Exp. Med. Apr., 1994, pp. 1145-1154. .
Pretolani et al., Antibody to Very Late Activation Antigen 4
Prevents Antigen-induced Bronchial Hyperreactivity and Cellular
Infiltration in the Guinea Pig Airways, J. Exp. Med., Sep. 1994,
pp. 795-805. .
Vanderslice et al., A Cyclic Hexapeptide Is a Potent Antagonist of
.alpha..sub.4 Integrins, J. Immunology, 185 (1997), pp. 1710-1718.
.
Yang, et al., "Interaction of Monocytoid Cels with the Mucosal
Addressin MAdCAM-1 via Integrins VAL-4 and LPAM-1." Abstract,
Immunol Cell Biol. 1996, 74(5), 383-393; abstract No.
1996:696526..
|
Primary Examiner: Carlson; Karen Cochrane
Assistant Examiner: Gupta; Anish
Attorney, Agent or Firm: Pillsbury Winthrop LLP
Claims
What is claimed is:
1. A cyclic peptide of the formula ##STR19##
where
Xaa.sub.1 is selected from an L-amino acid or a D-amino acid,
wherein the L-amino acid is selected from Phe, Lys and Arg, the
D-amino acid is selected from Phe and Met, and the L- or D-amino
acid optionally is substituted on its ax-carbon or ax-amino group
with a C.sub.1-4 alkyl group,
or
Xaa.sub.1 is MeIle;
Xaa.sub.2 is Leu, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.3 is Asp, optionally substituted on its .alpha.-carbon or
cc-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.4 is Val, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
X.sup.1 is a D-amino acid selected from Ala, Phe, Arg, Lys, Trp,
hArg(Et).sub.2, Orn(CHMe.sub.2), Orn(Me.sub.2), Lys(CHMe.sub.2) and
Arg(Pmc), optionally substituted on its .alpha.-carbon or its
.alpha.-amino group with a C.sub.1-4 alkyl group;
or
X.sup.1 is ##STR20##
NH(CH.sub.2).sub.5 CO, or NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO
where y is 1 or 2;
X.sup.2 is a D-amino acid selected from Ala, Arg, Lys, His,
hArg(Et).sub.2, Orn(CHMe.sub.2), and Orn(Me.sub.2), optionally
substituted on its .alpha.-carbon or its .alpha.-amino group with a
C.sub.1-4 alkyl group,
or
x.sup.2 is NH(CH.sub.2).sub.2 SCH.sub.2 CO, or NH(CH.sub.2).sub.x
CO where x is 2 or 3;
Xaa.sub.5 and Xaa.sub.6 are each, independently, a D-amino acid
selected from Ala and Arg, optionally substituted on its
.alpha.-carbon or .alpha.-amino group with a C.sub.1-4 alkyl
group;
p is 0 or 1; and
q is 0 or when p is 1, q is 0 or 1;
or a salt thereof;
with the proviso that when Xaa.sub.1 is MeIle, Xaa.sub.2 is Leu,
Xaa.sub.3 is Asp, Xaa.sub.4 is Val and p and q are both 0, then
i) X.sup.2 is not D-Ala, D-Arg, or D-Lys when X.sup.1 is D-Ala;
ii) X.sup.2 is not D-Arg when X.sup.1 is ##STR21##
iii) X.sup.2 is not D-Ala, D-Arg or D-His when X.sup.1 is
D-Arg;
iv) X.sup.2 is not D-Ala when X.sup.1 is D-Orn(CHMe.sub.2) or
D-Arg(Pmc);
v) x.sup.2 is not D-Ala or D-Lys when X.sup.1 is D-Lys; and
vi) X.sup.2 is not D-Lys or D-Arg when X.sup.1 is D-Phe or
D-Trp.
2. A cyclic peptide according to claim 1
where
Xaa.sub.1 is an L-amino acid selected from Meae, MePhe, Lys and
Arg, or a D-amino acid selected from Phe and Met;
Xaa.sub.2, Xaa.sub.3 and Xaa.sub.4 are, respectively, Leu, Asp, and
Val;
X.sup.1 is a D-amino acid selected from Ala, Phe, Arg, Lys, Trp,
hArg(Et).sub.2, Orn(CHMe.sub.2), Orn(Me.sub.2), Lys(CHMe.sub.2) and
Arg(Pmc),
or
X.sup.1 is ##STR22##
NH(CH.sub.2).sub.5 CO, or NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO
where y is 1 or 2;
X.sup.2 is a D-amino acid selected from Ala, Arg, Lys, His,
hArg(Et).sub.2, Orn(CHMe.sub.2), and Orn(Me.sub.2),
or
X.sup.2 is NH(CH.sub.2).sub.2 SCH.sub.2 CO, or NH(CH.sub.2).sub.x
CO where x is 2 or 3;
Xaa.sub.5 and Xaa.sub.6 are each, independently, a D-amino acid
selected from Ala and Arg;
or a salt thereof.
3. A cyclic peptide of the formula ##STR23##
where
Xaa.sub.1 is an L-amino acid selected from Phe, Lys and Arg, a
D-amino acid selected from Phe and Met, and the L- or D-amino acid
optionally is substituted on its .alpha.-carbon or its
.alpha.-amino group with a C.sub.1-4 alkyl group,
or
Xaa.sub.1 is MeIle;
Xaa.sub.2 is Leu, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.3 is Asp, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.4 is Val, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
X.sup.1 is a D-amino acid selected from Ala, Phe, Arg, Lys, Trp,
hArg(Et).sub.2, Orn(CHMe.sub.2), Orn(Me.sub.2), Lys(CHMe.sub.2) and
Arg(Pmc), optionally substituted on its .alpha.-carbon or its
.alpha.-amino group with a C.sub.1-4 alkyl group,
or
X.sup.1 is ##STR24##
NH(CH.sub.2).sub.5 CO, or NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO
where y is 1 or 2;
X.sup.2 is a D-amino acid selected from Ala, Arg, Lys, His,
hArg(Et).sub.2, Orn(CHMe.sub.2), and Orn(Me.sub.2), optionally
substituted on its o.alpha.-carbon or its .alpha.-amino group with
a C.sub.1-4 alkyl group,
or
X.sup.2 is NH(CH.sub.2).sub.2 SCH.sub.2 CO, or NH(CH.sub.2).sub.x
CO where x is 2 or 3;
Xaa.sub.5 and Xaa.sub.6 are each, independently, a D-amino acid
selected from Ala and Arg, optionally substituted on its
.alpha.-carbon or .alpha.-amino group with a C.sub.1-4 alkyl
group;
is 0 or 1; and
q is 0 or when p is 1, q is 0 or 1;
or a salt thereof,
with the proviso that when p and q are both 0, Xaa.sub.1 is not
MeIle.
4. A cyclic peptide of the formula ##STR25##
where
Xaa.sub.1 is a L-amino acid selected from MePhe and MeIle;
Xaa.sub.2 is Leu, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.3 is Asp, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.4 is Val, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
X.sup.1 is a D-amino acid selected from Ala and Arg, optionally
substituted on its .alpha.-carbon or its .alpha.-amino group with a
C.sub.1-4 alkyl group;
or
X.sup.1 is ##STR26##
or NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO where y is 1 or 2;
X.sup.2 is a D-amino acid selected from Ala and Arg, optionally
substituted on its .alpha.-carbon or its .alpha.-amino group with a
C.sub.1-4 alkyl group,
or
X.sup.2 NH(CH.sub.2).sub.x CO where x is 2 or 3;
Xaa.sub.5 and Xaa.sub.6 are each, independently, a D-amino acid
selected from Ala and Arg, optionally substituted on its
.alpha.-carbon or .alpha.-amino group with a C.sub.1-4 alkyl
group;
p is 1; and
q is 0 or 1;
or a salt thereof.
5. A cyclic peptide according to claim 1 where any two of X.sup.1,
X.sup.2, (Xaa.sub.5).sub.p and (Xaa.sub.6).sub.q are D-Arg or a
salt thereof.
6. A cyclic peptide according to claim 3 where Xaa.sub.1 is
MePhe.
7. A cyclic peptide according to claim 4 where p is 1 and q is 1;
or a salt thereof.
8. A cyclic peptide according to claim 4 where p is 1 and q is 0;
or a salt thereof.
9. A cyclic peptide according to claim 1 where the cyclic peptide
is selected from ##STR27##
c(MeIle-Leu-Asp-Val-D-Orn(CHMe.sub.2)-D-Orn(CHMe.sub.2))
c(MeIle-Leu-Asp-Val-D-Lys(CIHMe.sub.2)-D-Ala)
c(MeIle-Leu-Asp-Val-D-Orn(Me.sub.2)-D-Orn(Me.sub.2))
c(MeIle-Leu-Asp-Val-D-Ala-D-hArg(Et).sub.2)
c(MeIle-Leu-Asp-Val-D-Phe-D-hArg(Et).sub.2)
c(MeIle-Leu-Asp-Val-D-hArg(Et).sub.2 -D-hArg(Et).sub.2)
c(MeIle-Leu-Asp-Val-D-Lys-D-His)
c(Melle-Leu-Asp-Val-D-Arg(Pmc)-D-Lys)
c(MeIle-Leu-Asp-Val-D-Lys-D-Arg)
c(MePhe-Leu-Asp-Val-D-Ala-D-Lys)
c(MeIle-Leu-Asp-Val-D-Arg-D-Lys)
c(Lys-Leu-Asp-Val-D-Ala-D-Ala)
c(Arg-Leu-AspVal-D-Ala-D-Ala)
c(D-Phe-Leu-Asp-Val-D-Ala-D-Lys)
c(MePhe-Leu-Asp-Val-D-Ala-D-Ala)
c(D-Phe-Leu-Asp-Val-D-Ala-D-Lys)
c(D-Met-Leu-Asp-Val-D-Ala-D-Lys)
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg)
c(MePhe-Leu-Asp-Val-D-Arg-D-His)
c(MePhe-Leu-Asp-Val-D-Trp-D-Arg)
c(MePhe-Leu-Asp-Val-D-Arg-D-Ala-D-Arg)
c(MeIle-Leu-Asp-Val-D-Ala-D-Ala-D-Arg)
c(MeIle-Leu-Asp-Val-D-Arg-D-Ala-D-Arg)
c(MeIle-Leu-Asp-Val-D-Ala-D-Arg-D-Arg)
c(MePhe-Leu-Asp-Val-D-Ala-D-Arg-D-Arg)
c(Melhe-Leu-Asp-Val-D-Arg-D-Arg-D-Ala)
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg-D-Ala)
c(Melhe-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg)
c(MePhe-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg)
c(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Ala-D-Ala)
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.5 CO)
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.2 --S--(CH.sub.2).sub.2
--CO)
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.2 --S--CH.sub.2
--CO)
c(MeIle-Leu-Asp-Val-D-Arg-NH(CH.sub.2).sub.2 --S--CH.sub.2
--CO)
c(MeIle-Leu-Asp-Val-NH-CH.sub.2 --CH.sub.2 --S--CH.sub.2
--CO-D-Arg-D-Arg), and
c(MePhe-Leu-Asp-Val-NH-CH.sub.2 --CH.sub.2 --S--CH.sub.2
--CO-D-Arg-D-Arg).
10. A cyclic peptide according to claim 9 selected from
c(MeIle-Leu-Asp-Val-D-hArg(Et).sub.2 -D-hArg(Et).sub.2)
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg)
c(MePhe-Leu-Asp-Val-D-Arg-D-His)
c(MePhe-Leu-Asp-Val-D-Trp-D-Arg)
c(MePhe-Leu-Asp-Val-D-Arg-D-Ala-D-Arg)
c(MeIle-Leu-Asp-Val-D-Arg-D-Ala-D-Arg)
c(MeIle-Leu-Asp-Val-D-Ala-D-Arg-D-Arg)
c(MePhe-Leu-Asp-Val-D-Ala-D-Arg--D-Arg)
c(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Ala)
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg-D-Ala)
c(MeIle-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg)
c(MePhe-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg)
c(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Ala-D-Ala)
c(MeIle-Leu-Asp-Val-NH-CH.sub.2 --CH.sub.2 --S--CH.sub.2
--CO-D-Arg-D-Arg), and
c(MePhe-Leu-Asp-Val-NH-CH.sub.2 --CH.sub.2 --S--CH.sub.2
--CO-D-Arg-D-Arg).
11. A cyclic peptide according to claim 10 of formula
c(MePhe-Leu-Asp-Val-D-Arg-D-His).
12. A cyclic peptide according to claim 10 of formula
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg).
13. A cyclic peptide according to claim 10 of formula
c(MePhe-Leu-Asp-Val-D-Ala-D-Arg-D-Arg).
14. A cyclic peptide according to claim 10 of formula
c(MePhe-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg).
15. A pharmaceutical composition comprising a cyclic peptide
according to any one of claims 1, 2, 5, 3, 6, 4 and 7 to 14, or and
7 to 14, or a pharmaceutically acceptable salt thereof in
association with a pharmaceutically acceptable diluent or
carrier.
16. A method for inhibiting the interaction between VCAM-1 and/or
fibronectin and the integrin receptor VLA-4 in mammals in need of
such treatment which comprises administering to said mammal an
effective amount of a cyclic peptide according to any one of claims
1, 2, 5, 3, 6, 4 and 7 to 14, or a pharmaceutically acceptable salt
thereof.
17. The method according to claim 16 for treating multiple
sclerosis, rheumatoid arthritis, asthma or psoriasis.
18. A method for inhibiting the interaction between MAdCAM-1 and
the integrin .alpha.4.beta.7 in mammals in need of such treatment
which comprises administering to said mammal an effective amount of
a cyclic peptide according to any one of claims 1, 2, 5, 3, 6, 4
and 7 to 14, or a pharmaceutically acceptable salt thereof.
19. A process for the manufacture of a cyclic peptide or a salt
thereof as claimed in any of claims 1, 2, 5, 3, 4, and 7 to 14,
selected from process routes (a), (b) and (c):
(a) assembling the required linear peptide in a stepwise manner
(adding one amino acid at a time) followed by selective removal of
any N- and C-terminal protecting groups, cyclisation and finally
deprotonation to give said cyclic peptide, and optionally
converting said cyclic peptide into a salt thereof;
(b) forming an amide bond by coupling two peptide units, one
containing a carboxylic acid group, or a reactive derivative
thereof, and the other containing an amino group, such that said
cyclic peptide in protected or unprotected form is produced, and
removing any protecting group using process route (c) below, and
optionally converting the product thus obtained into a salt
thereof; and
(c) removing one or more conventional peptide protecting groups
from a protected cyclic peptide of formula ##STR28##
where Pr.sup.1 is a protecting group on the acid group in the side
of chain of Xaa.sub.3 to give said cyclic peptide in protected or
unprotected form, simultaneously or subsequently removing any
additional conventional peptide protecting group present, and
optionally converting the product thus obtained into a salt
thereof.
Description
Many of the cell-cell and cell-extracellular matrix interactions
are mediated by protein ligands (e.g. fibronectin, vitronfectin and
VCAM-1) and their integrin receptors [e.g. VLA-4
(.alpha.4.beta.1)]. Recent studies have shown these interactions to
play an important role in many physiological (e.g. embryonic
development and wound healing) and pathological (e.g. tumour-cell
invasion and metastasis, inflammation, atherosclerosis and
autoimmune diseases) conditions. Agents which can selectively
inhibit some of these interactions are predictably useful for the
treatment of a number of diseases.
Integrins are heterodimeric cell surface receptors that are
composed of noncovalently associated .alpha. and .beta. subunits.
Using molecular biology and protein chemistry, a number of .alpha.
and .beta. subunits have been identified. The integrin family can
be subdivided into classes based on the .beta. subunits, which can
be associated with one or more .alpha. subunits. The most widely
distributed integrins belong to the .beta.1 class, also known as
the very late antigens (VLA). The second class of integrins are
leukocyte-specific receptors and consist of one of three a subunits
(.alpha.L, .alpha.M, or .alpha.X) complexed with the .beta.2
protein. The cytoadhesins .alpha.IIb.beta.3 and .alpha.v.beta.3,
constitute a third class of integrins. A fourth class of integrins
includes .alpha.4.beta.7.
A wide variety of proteins serve as ligands for integrin receptors.
In general, the proteins recognised by integrins fall into one of
three classes: extracellular matrix proteins, plasma proteins, and
cell surface molecules. Extracellular matrix proteins such as
collagen, fibronectin, fibrinogen, laminin, thrombospondin, and
vitronectin bind to a number of integrins. Many of these adhesive
proteins also circulate in plasma and bind to activated blood
cells. Additional components in plasma that are ligands for
integrins include fibrinogen and factor X. Cell-bound complement
C3bi and several transmembrane proteins, such as Ig-like cell
adhesion molecule (ICAM-1,2,3) and vascular cell adhesion molecule
(VCAM-1), which are members of the Ig superfamily, also serve as
cell-surface ligands for some integrins. Mucosal addressin cell
adhesion molecule-1 (MAdCAM-1) is another member of the Ig
superfamily and is bound by the integrin .alpha.4.beta.7.
The target amino acid sequences for many integrins have been
identified. For example, the target sequence in .alpha.5.beta.1,
.alpha.II.beta.3, and .alpha.v.beta.3, is the Arg-Gly-Asp
tripeptide found in proteins such as fibronectin, fibrinogen,
thrombospondin, type 1 collagen, vitronectin and vWF. However, the
Arg-Gly-Asp sequence is not the only integrin recognition motif
used by adhesive ligands. Another integrin .alpha.4.beta.1 binds
the variable region (CS1) of fibronectin via the sequence
Leu-Asp-Val and the platelet integrin .alpha.IIb.beta.3 also
recognises the sequence
His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val at the
carboxy-terminus of the gamma chain of fibrinogen.
The present invention principally relates to agents which block the
interaction of the ligand VCAM-1 to its integrin receptor VLA-4
(.alpha.4.beta.1). [Reference for a review on VLA-4: Structure of
the Integrin VLA-4 and Its Cell--Cell and Cell Matrix Adhesion
Functions, M. E. Hemler, M. J. Elices, C. Parker and Y. Takada,
Immunological Reviews, 114 (1990) 45-65.] Integrin .alpha.4.beta.1
is expressed on numerous hematopoietic cells and established cell
lines, including hematopoietic precursors, peripheral and cytotoxic
T lymphocytes, B lymphocytes, monocytes, thymocytes and
eosinophils. Unlike other .beta.1 integrins that are involved only
in cell-extracellular matrix interactions, .alpha.4.beta.1 mediates
both cell--cell and cell-extracellular matrix interactions. Cells
expressing activated .alpha.4.beta.1 bind to the carboxy-terminal
cell binding domain of fibronectin (non Arg-Gly-Asp mediated), to
VCAM-1 expressed on endothelial cells, and to each other to promote
homotypic aggregation. The expression of VCAM-1 by endothelial
cells is upregulated by proinflammatory cytokines such as
INF-.gamma., TNF-.alpha.and IL-1.beta..
The present invention also relates to agents which block the
interaction of the ligand MAdCAM-1 and the integrin
.alpha.4.beta.7.
Regulation of .alpha.4.beta.1-mediated cell adhesion is important
in numerous physiologic processes, including T-cell proliferation,
B-cell localisation to germinal centres, and adhesion of activated
T cells and eosinophils to endothelial cells. In addition, integrin
.alpha.4.beta.1-mediated processes have been implicated in tumour
cell metastasis and diseases involving lymphocyte, monocyte or
eosinophil recruitment such as multiple sclerosis, rheumatoid
arthritis, asthma, psoriasis, insulin-dependent diabetes,
glomerulonephritis, inflammatory bowel disease, ischaemic heart
disease, myocarditis, peripheral vascular disease, transplant
rejection, for example chronic allograft rejection, and allergic
disorders. Evidence for the involvement of VLA-4VCAM-1 interaction
in the above disease processes has been accumulated by
investigating the role of the peptide CS-1 and antibodies specific
for VLA-4 or VCAM-1 in various in vitro and in vivo experimental
models of inflammation (e.g. contact cutaneous hypersensitivity
response in mice), experimental autoimmune encephalomyelitis, lung
antigen challenge, diabetes, ulcerative colitis, nephritis and
allograft rejection. Additionally, integrin
.alpha.4.beta.7-mediated processes have been implicated in
lymphocyte recruitment in diseases such as inflammatory bowel
disease and insulin-dependent diabetes.
For example, in an experimental model of arthritis (arthritis
induced in inbred female Lewis rats with a single intraperitoneal
injection of peptidoglycan-polysaccharide fragments from group A
streptococcal cell walls), intravenous administration of CS-1 at
the initiation of arthritis (days 0-4; 300 .mu.g/day) or on days 11
to 16 in animals with established arthritis, was shown to suppress
both acute and chronic inflammation. [Reference: Synthetic
Fibronectin Peptides Suppress Arthritis in Rats by Interrupting
Leukocyte Adhesion and Recruitment, S. M. Wahl, J. B. Allen, K. L.
Hines, T. Imamichi, A. M. Wahl, L. T. Furcht and J. B. McCarthy, J.
Clin. Invest, 94 (1994) 655-662].
In another model of inflammation (contact hypersensitivity response
in oxazalone or 2.4-dinitrofluorobenzene-sensitised mice),
intravenous administration of the anti-.alpha.-4 specific
monoclonal antibodies R1-2 or PS/2 (4 to 6 hours prior to
challenge) significantly inhibited (50-60% reduction in the ear
swelling response) the efferent response. [Reference:
Monoclonal Antibodies to the Integrin .alpha.-4 Subunit Inhibit the
Murine Contact Hypersensitivity Response, P. L. Chisholm, C. A.
Williams and R. R. Lobb, Eur. J. Immunol., 23 (1993) 682-688]. In
an intestinal inflammation model (acute colitis in Cotton-top
tamarin), anti-.alpha.4 integrin monoclonal antibody HP1/2 that
binds VLA4 resulted in significant attenuation of acute colitis. In
contrast, two anti-E-selectin monoclonal antibodies (BB11 and EH8)
slightly diminished colitis after the 10-day treatment period in
Cotton-top tamarin [Reference: Attenuation of Colitis in the
Cotton-top Tamarin by Anti-.alpha. 4 Integrin Monoclonal Antibody,
D. K. Podolsky, R. Lobb, N. King, C. D. Benjamin, B. Pepinsky, P.
Sehgal and M. deBeaumont, J. Clin. Invest., 92 (1993) 372-380].
The antibodies have also been shown to be effective in a model of
autoimmune encephalomyelitis (EAE). EAE is an inflammatory
condition of the central nervous system with similarities to
multiple sclerosis. In the model the inflammation is induced
experimentally. In both EAE and multiple sclerosis, circulating
leukocytes penetrate the blood-brain barrier and damage myelin,
resulting in impaired nerve conduction and paralysis. EAE can be
induced actively by priming an animal to CNS proteins like myelin
basic protein (MBP), or adoptively by injection of activated
lymphocytes that are specific for these CNS antigens. Various
monoclonal antibodies, MK/1 (anti-VCAM-1) and PS/2 and LPAM-1 (anti
.alpha.4 integrin), when injected into irradiated female
(PL/IJ.times.SJL)F1 mice delayed the onset of disease. When
injection of antibody to a.sup.4 integrin (i.e. LPAM-1 and PS/2)
was continued every 3 days until after onset of disease, not only
was the onset of disease delayed, but in this case severity of
disease was also significantly decreased. [Reference: Surface
Expression of .alpha. 4 Integrin by CD4 T Cells Is Required for
Their Entry into Brain Parenchyma, J. L. Baron, J. A. Madri, N. H.
Ruddle, J. Hashim and C. A. Janeway, Jr., J. Exp. Med., 177 (1993)
57-68].
Monoclonal antibodies to VCAM-1 (M/K-1) and VLAA (PS-2) have also
been shown to be active in experimental asthma models
(antigen-induced eosinophil and T-cell recruitment). Both these
antibodies when injected intraperitoneally 24 hours before the
inhaled ovalbumin challenge significantly decreased (73-74%)
eosinophil infiltration in mice [Reference: Role of vascular cell
adhesion molecule-1/very late activation antigen 4 and
intracellular adhesion molecule 1/lymphocyte function-associated
antigen 1 interactions in antigen-induced eosinophil and
T-recruitment into the tissue, H. Nakajima, H. Sano, T. Nishimura,
S. Yoshida and I. Iwamoto, J Exp. Med., 179 (1994) 1145-1154].
Similar results were obtained in guinea pigs when anti-VLA-4
monoclonal antibody HP1/2 was injected (3-10 mg/kg) before
ovalbumin challenge [Reference: Antibody to very late activation
antigen 4 prevent antigen -induced bronchial hyperreactivity and
cellular infiltration in the guinea-pig airways, M. Pretolani, C.
Ruffie, J-R Lapa e Silva, D. Joseph, R. R. Lobb and B. B.
Vargaftig, J. Exp. Med., 180 (1994), 795-805 and Role of the VLA-4
integrin in leukocyte recruitment and bronchial hyperresponsiveness
in the guinea-pig, A. A. Y. Milne and P. J. Piper, European J
Pharmacol., 282 (1995), 243-249].
Antibodies specific for both .alpha.4-integrin (LPAM-1) and one of
its ligands, VCAM-1, were also shown to be effective in treating
insulin-dependent diabetes mellitus in the nonobese diabetic mouse.
Insulin-dependent diabetes mellitus is believed to be an autoimmune
disease in which activated T lymphocytes destroy the
insulin-producing .beta.-cells of the pancreatic islets. The
antibody R1-2 prevented the onset of insulitis in a dose-dependent
manner in nonobese diabetic mice. The blocking of disease was
accompanied by a marked decrease in lymphocytic infiltration of the
islets of Langerhans. [Reference: The Pathogenesis of Adoptive
Murine Autoimmune Diabetes Requires an Interaction Between .alpha.
4-Integrins and Vascular Cell Adhesion Molecule-1, J. L. Baron,
E-P. Reich, I. Visintin and C. A. Janeway, Jr., J. Clin. Invest.,
93 (1994) 1700-1708].
Cells expressing integrin .alpha.4.beta.1 have been shown to bind
to sequences in the heparin II binding domain and the alternatively
spliced type III connecting segment (IIICS) located in the
carboxy-terminal cell binding domain of fibronectin. Within the
IIICS region, .alpha.4.beta.1 binds with high affinity to a
peptirde sequence termed CS-1 (a 25-amino acid peptide), suggesting
that this is the major site of .alpha.4.beta.1 interaction in
fibronectin. The tripeptide Leu-Asp-Val is the minimal sequence
within CS-1 capable of supporting hematopoietic cell adhesion or of
inhibiting .alpha.4.beta.1-mediated cell binding to fibronectin.
[References for CS1: The Minimal Essential Sequence for a Major
Cell Type-Specific Adhesion Site (CS1) Within the Alternatively
Spliced Type III Connecting Segment Domain of Fibronectin is
Leucine-Aspartic Acid-Valine, A. Komoriya, L. J. Green, M. Mervic,
S. S. Yamada, K. M. Yamada and M. J. Humphries, J. Biol. Chem., 23
(1991) 15075-15079; Activation-Dependent Recognition by
Hematopoietic Cells of the LDV Sequence in the V Region of
Fibronectin, E. A. Wayner and N. L. Kovach, J. Cell Biol., 116
(1992) 489-497.]
In addition to the Leu-Asp-Val containing sequences mentioned
above, a cyclic octapeptide
1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (containing a
disulphide bridge between the two cysteine residues) has been
reported to be as effective as the LDV containing peptide
Cys-Leu-His-Gly-Pro-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr in blocking
Jurkat cell adhesion to CS-1 coated plates (IC.sub.50 30 .mu.M).
The cyclic peptide also inhibited the binding of Jurkat cells to
fibronectin coated plates. In addition to inhibiting
.alpha.4.beta.1-induced adhesion, the octapeptide also inhibited
function in .alpha.v.beta.3 as well as
.alpha.IIb.beta.IIIa-dependent assays. Therefore the peptide is not
selective for .alpha.4.beta.1-mediated adhesion. [Reference: Cyclic
RGD Peptide Inhibits .alpha.4.beta.1 Interaction with Connecting
Fragment 1 and Vascular Cell Adhesion Molecule, P. M. Cardarelli,
R. R. Cobb, D. M. Nowlin, W. Scholz, F. Gorcsan, M. Moscinski, M.
Yasuhara, S-L. Chiang and T. J. Lobl, J. Biol. Chem., 269 (1994)
18668-18673.]
A few small non-peptidic compounds [Reference: Non-peptidic
Surrogates of the Leu-Asp-Val Sequence and Their Use in the
Treatrnent of Inflammation, Autoimmune Diseases and Tumour
progression, YEDA Research and Development Co. Ltd, WO 94/02445,
Publ. date Feb. 3, 1994] have also been reported to inhibit
.alpha.4.beta.1-induced adhesion.
Furthermore, in WO 96/00581, Publi. date Jan. 11, 1996, a number of
cyclic peptides containing the Leu-Asp-Val sequence and at least
one Cys amino acid residue and/or a Glu amino acid residue have
been reported to inhibit the binding of .alpha.4.beta.1integrin to
VCAM-1 or fibronectin. However, three of the cyclic peptides
disclosed in this patent application (namely,
c(Glu-TripLeu-Asp-Val), c(Glu-Trp-Leu-Asp-Val-Asp) and
c(Glu-Trp-Leu-Asp-Val-Pro-Glu-Trp-Leu-Asp-Val), where c indicates
the amino acid sequence is cyclised), were shown to te very poor
inhibitors of the binding of HL-60 cells to VCAM-1-IgG fusion
protein (IC.sub.50 values of 94,>1000 and>1000 .mu.M
respectively). [Reference: P. Vanderslice, K. Ren, J. K. Revelle,
D. C. Kim, D. Scott, R. J. Bjercke, E. T. H. Yeh, P. J. Beck and T.
P. Kogan, J. Immunology, 158, (1997), 1710-1718].
A disulphide cyclic pentapeptide, Arg-Cys-Asp-thioproline-Cys
(thioproline=thiazolidine-4-carboxylic acid), has also been
reported to be an inhibitor of leukocyte cell adhesion to
fibronectin. In addition, the cyclic peptide also inhibited the
binding to the 120 kDa chymotryptic fragment of fibronectin, which
contains the Arg-Gly-Asp central cell binding domain. Again, the
peptide was not selective binding to both .alpha.4.beta.1 and
.alpha.5.beta.1 [Reference: A Novel Cyclic Pentapeptide Inhibits
.alpha.4.beta.1 and .alpha.5.beta.1 Integrin-Mediated Cell
Adhesion, D. M. Nowlin, F. Gorcsan, M. Moscinski, S-L. Chiang, T.
J. Lobl and P. M. Cardarelli, J. Biol. Chem., 268 (1993)
20352-20359.]
In our copending PCT application, WO96/20216, Publi. date Jul. 4,
1996, cyclic peptides containing the Leu-Asp-Val sequence which
inhibit the binding of .alpha.4.beta.1 integrin to VCAM-1 or
fibronectin are reported.
Although a number of peptides that inhibit the interaction of
VCAM-1 and fibronectin with integrin VLA4 have been discovered,
there is a continuing need for alternative compounds which inhibit
this interaction and, in particular, for compounds which can be
formulated into slow release pharmaceutical compositions. There is
also a need for compounds which inhibit the interaction of MAdCAM-1
with the integrin .alpha.4.beta.7.
According to one aspect of the present invention there is provided
a cyclic peptide of formula ##STR3##
where
Xaa.sub.1 is selected from L-amino acids selected from Phe, Lys and
Arg; D-amino acids selected from Phe and Met, the L- and D-amino
acid optionally substituted on its .alpha.-carbon or its
.alpha.-amino group with a C.sub.1-4 alkyl group; and MeIle;
Xaa.sub.2 is Leu, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group; Xaa.sub.3 is Asp,
optionally substituted on its .alpha.-carbon or .alpha.-amino group
with a C.sub.1-4 alkyl group; Xaa.sub.4 is Val, optionally
substituted on its .alpha.-carbon or .alpha.-amino group with a
C.sub.1-4 alkyl group; X.sup.1 is selected from D-amino acids
selected from Ala, Phe, Arg, Lys, Trp, hArg(Et).sub.2,
Orn(CHMe.sub.2), Orn(Me.sub.2), Lys(CHMe.sub.2) and Arg(Pmc),
optionally substituted on their .alpha.-carbon or their
.alpha.-amino group with a C.sub.1-4 alkyl group; ##STR4##
NH(CH.sub.2).sub.5 CO; and NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO,
where y is 1 or 2;
X.sup.2 is selected from D-amino acids selected from Ala, Arg, Lys,
His, hArg(Et).sub.2, Orn(CHMe.sub.2), and Om(Me.sub.2), optionally
substituted on their .alpha.-carbon or their .alpha.-amino group
with a C.sub.14 alkyl group; NH(CH.sub.2).sub.2 SCH.sub.2 CO; and
NH(CH.sub.2).sub.x CO, where x is 2 or 3;
Xaa.sub.5 and Xaa.sub.6 are each, independently a D-amino acid
selected from Ala and Arg, optionally substituted on its
.alpha.-carbon or .alpha.-amino group with a C.sub.1-4 alkyl
group;
P is 0 or 1; ;and q is 0 or when p is 1,q is 0 or 1;
or a salt thereof;
with the proviso that when Xaa.sub.1 is MeIle, Xaa.sub.2 is Leu,
Xaa.sub.3 is Asp, Xaa.sub.4 is Val and p and q are both 0,
i) X.sup.2 is not D-Ala, D-Arg, or D-Lys when X.sup.1 is D-Ala;
ii) x.sup.2 is not D-Arg when X.sup.1 is ##STR5##
iii) X.sup.2 is not D-Ala, D-Arg or D-His when X.sup.1 is
D-Arg;
iv) X.sup.2 is not D-Ala when X.sup.1 is D-Orn (CHMe.sub.2) or
D-Arg(Pmc);
v) X.sup.2 is not D-Ala or D-Lys when X.sup.1 is D-Lys; and
vi) X.sup.2 is not D-Lys or D-Arg when X.sup.1 is D-Phe or
D-Trp.
The cyclic peptides preferably have an IC.sub.50 of <10 .mu.M,
more preferably <5 .mu.M in the MOLT-4 cell-fibronectin assay
hereinafter described or an IC.sub.50 <30 .mu.M, more preferably
<5 .mu.M in the MOLT-4 cell/recombinant soluble VCAM-1 assay
hereinafter described.
The cyclic peptides are also active in the JY cell/MAdCAM-1
Adhesion assay, hereinafter described.
It is to be understood that amino acids have the L-configuration
unless otherwise stated and that all residue components, that is
Xaa.sub.1 to Xaa.sub.6 X.sup.1 and X.sup.2 forming the cyclic
peptides are written left to right in the direction from the amino
(N-terminus) to the carboxyl (C-terminus) groups. The residue
components are linked together so that the carboxyl terminus of one
residue is linked to the amino terminus of the adjacent residue.
Where a naturally occurring amino acid has more than one carboxyl
and/or amino group, linking is respectively through the ac-carboxyl
and the .alpha.-amino groups. Linking for the residues
D-hArg(Et).sub.2, D-Orn(CHMe.sub.2), D-Orn(Me.sub.2),
D-Lys(CHMe.sub.2) and D-Arg(Pmc) is hereinafter defined with
respect to the figures. The naturally occurring amino acids
residues in the cyclic peptides are generally defined in terms of a
three letter code where Ala is alanine, His is histidine, Ile is
isoleucine, Lys is lysine, Arg is arginine, Val is valine, Asp is
aspartic acid, Met is methionine, Phe is phenylalanine, Leu is
leucine, and Trp is tryptophan. MePhe and MeIle respectively refer
to N-methyl phenyalanine and N-methyl isoleucine. D-hArg(Et).sub.2,
D-Orn(CHMe.sub.2), D-Orn(Me.sub.2), D-Lys(CHMe.sub.2) and
D-Arg(Pmc) are hereinafter defined in the figures.
It is to be understood that generic terms such as `alkyl` include
both straight and branched chain variants. Compounds of the present
invention include solvates such as for example hydrates and include
prodrugs such as, for example, in vivo hydrolysable esters.
Preferably Xaa.sub.1 includes L-MeIle, L-MePhe, L-Lys, L-Arg, D-Phe
and D-Met. Preferred values for Xaa.sub.2, Xaa.sub.3 and Xaa.sub.4
are respectively Leu, Asp, and Val. X.sup.1 is preferably selected
from D-amino acids selected from Ala, Phe, Arg, Lys, Trp,
hArg(Et).sub.2, Orn(CHMe.sub.2), Orn(Me.sub.2), Lys(CHMe.sub.2) and
Arg(Pmc); and ##STR6##
NH(CH.sub.2).sub.5 CO; and NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO,
where y is 1 or 2. x.sup.2 is preferably selected from D-amino
acids selected from Ala, Arg, Lys, His, hArg(Et).sub.2,
Orn(CHMe.sub.2), and Orn(Me.sub.2); NH(CH.sub.2).sub.2 SCH.sub.2
CO, and NH(CH.sub.2).sub.x CO, where x is 2 or 3. Xaa.sub.5 and
Xaa.sub.6 are preferably each, independently D-Ala or D-Arg.
Cyclic peptides or salts thereof in which any two of X.sup.1,
X.sup.2, (Xaa.sub.5).sub.p and (Xaa.sub.6).sub.q are D-Arg
represent a particularly preferred aspect of the invention.
According to a further aspect of the invention, cyclic peptides
have the formula formula ##STR7##
where
Xaa.sub.1 is selected from L-amino acids selected from Phe, Lys and
Arg; D-amino acids selected from Phe and Met, the L- and D-amino
acid optionally substituted on its .alpha.-carbon or its
.alpha.-amino group with a C.sub.1-4 alkyl group; and MeIle; and
most preferably is MePhe;
Xaa.sub.2 is Leu, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.3 is Asp, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.4 is Val, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
X.sup.1 is selected from D-amino acids selected from Ala, Phe, Arg,
Lys and Trp, optionally substituted on their .alpha.-carbon or
their .alpha.-amino group with a C.sub.1-4 alkyl group,
hArg(Et).sub.2, Orn(CHMe.sub.2), OM(Me.sub.2), Lys(CHMe.sub.2) and
Arg(Pmc); and ##STR8##
NH(CH.sub.2).sub.5 CO; and NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO,
where y is 1 or 2;
X.sup.2 is selected from D-amino acids selected from Ala, Arg, Lys
and His, optionally substituted on their .alpha.-carbon or their
.alpha.-amino group with a C.sub.1-4 alkyl group, hArg(Et).sub.2,
Orn(CHMe.sub.2), and Orn(Me.sub.2); NH(CH.sub.2).sub.2 SCH.sub.2
CO; and NH(CH.sub.2).sub.x CO, where x is 2 or 3;
Xaa.sub.5 and Xaa.sub.6 are each, independently a D-amino acid
selected from Ala and Arg, optionally substituted on its
.alpha.-carbon or .alpha.-amino group with a C.sub.1-4 alkyl
group;
p is 0 or 1; and q is 0 or when p is 1, q is 0 or 1; or a salt
thereof; with the proviso that when p and q are both 0, Xaa.sub.1
is not MeIle; or a salt thereof In such a cyclic peptide when
Xaa.sub.1 is MeIle, p is 1 and q is either 0 or 1.
According to yet a further aspect of the invention cyclic peptides
have the formula ##STR9##
where
Xaa.sub.1 is a L-amino acid selected from MePhe and MeIle;
Xaa.sub.2 is Leu, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.3 is Asp, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
Xaa.sub.4 is Val, optionally substituted on its .alpha.-carbon or
.alpha.-amino group with a C.sub.1-4 alkyl group;
X.sup.1 is selected from D-amino acids selected from Ala and Arg,
optionally substituted on their .alpha.-carbon or their
.alpha.-amino group with a C.sub.1-4 alkyl group; ##STR10##
and NH(CH.sub.2).sub.2 S(CH.sub.2).sub.y CO, where y is 1 or 2;
X.sup.2 is selected from D-amino acids selected from Ala and Arg
optionally substituted on their .alpha.-carbon or their
.alpha.-amino group with a C.sub.1-4 alkyl group; and
NH(CH.sub.2).sub.x CO, where x is 2 or 3;
Xaa.sub.5 and Xaa.sub.6 are each, independently a D-amino acid
selected from Ala and Arg, optionally substituted on its
.alpha.-carbon or .alpha.-amino group with a C.sub.1-4 alkyl
group;
p is 1;and q is 0 or 1;
or a salt thereof.
Particularly preferred cyclic peptides according to the invention
are cyclic peptides having any of the structures of compounds 1-38
in Table 2 hereinafter; or a salt thereof. Any amino acid in
structures 1-38 is optionally substituted on its .alpha.-carbon or
.alpha.-amino group with C.sub.1-4 alkyl (especially methyl).
Preferred cyclic peptides are any of compounds 8, 20-23, 25-32, 37
and 38. More preferred cyclic peptides are any of compounds 20, 21,
27 and 31.
The cyclic peptides of the present invention have at least one of
the following advantages: they are more potent than known
compounds, e.g. CS-1 (a 25-amino acid peptide) in our tests; they
are smaller than CS-1, and therefore easier to synthesise, and
being cyclic are more stable to enzymic degradation; and they are
compatible with slow release pharmaceutical compositions.
Preferred compounds have shown activity in a number of in vivo
screens in mice, for example, delayed-type hypersensitivity
responses induced by oxazolone in the skin (contact
hypersensitivity, CHS, or by ovalbumin in the footpad (delayed-type
hypersensitivity, DTH), collagen-induced arthritis, antigen-induced
bronchiolar lavage eosinophilia and experimental allergic
encepholmyelitis. For example, the dose of CS-1 required to produce
a half-maximal inhibitory response in DTH was 1 mg/kg/day while
that for compound 20 was 0.01 mg/kg/day. No toxicity at the
effective dose was observed for the compounds of the present
invention.
According to a further feature of the invention there is provided a
pharmaceutical composition which comprises a cyclic peptide of the
invention in association with a pharmaceutically acceptable diluent
or carrier. The invention also provides a pharmaceutical
composition which comprises a pharmaceutically acceptable salt of a
cyclic peptide according to the invention in association with a
pharmaceutically acceptable diluent or carrier.
Pharmaceutically acceptable salts include, for example, for cyclic
peptides that are sufficiently basic, for example, those having a
free guanidino group such as arginine, salts with acids forming
physiologically acceptable anions, such as salts with mineral
acids, for example, hydrogen halides such as hydrogen chloride and
hydrogen bromide, sulphonic and phosphonic acids; and with organic
acids, especially acetic, oxalic, tartaric, mandelic,
p-toluenesulphonic, methanesulphonic acids and the like. For cyclic
peptides which are sufficiently acidic, for example those having a
free carboxylic acid group, salts with bases forming
physiologically acceptable cations such as salts with alkali metal
such as sodium and potassium; alkaline earth metals such as
magnesium and calcium; aluminium and ammonium salts, and salts with
organic bases such as ethanolamine, methylamine, diethylamine,
isopropylamine, trimethylamine and the like. Such salts may be
prepared by any suitable method known in the art.
The composition may be in a form suitable for oral use, for example
a tablet, capsule, aqueous or oily solution, suspension or
emulsion; for nasal use, for example a snuff, nasal spray or nasal
drops; for vaginal or rectal use, for example a suppository; for
administration by inhalation, for example as a finely divided
powder or a liquid aerosol; for sub-lingual or buccal use, for
example a tablet or capsule; or for parenteral use (including
intravenous, subcutaneous, intramuscular, intravascular or
infusion), for example a sterile aqueous or oily solution or
suspension, or a depot formulation with drug incorporated in a
biodegradable polymer. The composition may be in a form suitable
for topical administration such as for example creams, ointments
and gels. Skin patches are also contemplated. Formulation in
general is described in Chapter 25.2 of Comprehensive Medicinal
Chemistry, Volume 5, Editor Hansch et al, Pergamon Press 1990.
In general the above compositions may be prepared in a conventional
manner using conventional excipients. However, in the case of a
composition for oral administration, it may be convenient for the
composition to include a coating to protect the cyclic peptide
active ingredient from the actions of enzymes in the stomach.
A preferred composition of the invention is one suitable for oral
administration in unit dosage form for example a tablet or capsule.
Which contains from 2.5 to 500 mg, and preferably 10 to 100 mg, of
cyclic peptide in each unit dose, or one suitable for parenteral
administration which contains from 0.5 to 100 mg of cyclic peptide
per ml, and preferably 1 to 10 mg of cyclic peptide per ml of
solution.
A parenteral composition is preferably a solution in isotonic
saline or isotonic dextrose buffered if necessary to a pH of 5 to
9. Alternatively, the parenteral composition may be one designed
for slow release in which case the amount of cyclic peptide per
unit dose is in general greater than that required when a
conventional injectable formulation is used. A preferred slow
release formulation is a continuous release formulation, for
example a formulation of the type described in European Patent
Specification No. 58481. For slow release formulations containing
polylactic/polyglycolic based polymers it is preferred that the
cyclic peptide of the invention contains a basic group. For such
cyclic peptides an alternative slow release formulation is as
described in International Patent Application, Publication No.
WO93/24150. A basic amino acid is defined as one containing a basic
functional group in its side chain, such as for example amino or
guanidino either of which may be optionally substituted with
C.sub.1-4 alkyl. One such particularly preferred basic amino acid
is arginine. In a particularly preferred embodiment of the
invention in the cyclic peptide of formula I any two of X.sup.1,
X.sup.2, (Xaa.sub.5).sub.q and (Xaa.sub.6 ).sub.q are D-Arg. Such
cyclic peptides containing a basic amino acid can form so-called
`peptide-polymer` salts with acid ended polyesters, for example
polylactides. These salts have advantageous solubility
characteristics which makes them particularly suitable for
manufacturing slow release parenteral formulations which have
beneficial release profiles and are stable on storage. A preferred
slow release parenteral formulation contains from 1 to 100 mg of
cyclic peptide per unit dose. A preferred pharmaceutical
composition is for parenteral administration designed for slow
release over a period of at least 5 days.
The composition of the invention will normally be administered such
that a daily oral dose will be from 0.1 mg/kg, to 50 mg/kg and a
daily parenteral dose, will be from 5, preferably from 20,
micrograms/kg to 10 mg/kg.
According to a further feature of the invention there is provided a
method for inhibiting the interaction between VCAM-1 and/or
fibronectin and the integrin receptor VLA-4 in warm-blooded
mammals, such as man, in need of such treatment which comprises
administering to said animal an effective amount of a cyclic
peptide of the invention or a pharmaceutically acceptable salt
thereof. The invention also provides the use of such a cyclic
peptide or a pharmaceutically-acceptable salt thereof in the
production of a new medicament for use in the treatment of a
disease ominedical condition mediated by the interaction between
fibronectin and/or VCAM-1 (especially VCAM-1) and the integrin
receptor VLA4. Utility as tools for research is also
contemplated.
According to a further aspect of the invention there is provided a
method for inhibiting the interaction between MAdCAM-1 and the
integrin .alpha.4.beta.7 in warm-blooded mammals, such as man, in
need of such treatment which comprises administering to said animal
an effective amount of a cyclic peptide of the invention or a
pharmaceutically acceptable salt thereof. The invention also
provides the use of such a cyclic peptide or a
pharmaceutically-acceptable salt thereof in the production of a new
medicament for use in the treatment of a disease or medical
condition mediated by the interaction between MAdCAM-1 and the
integrin .alpha.4.beta.7. Utility as tools for research is also
contemplated.
According to another aspect of the present invention there is
provided a cyclic peptide of the invention as herein described for
use as a medicament. According to another aspect of the present
invention there is provided a method for inhibiting the interaction
between VCAM-1 and/or fibronectin and the integrin receptor VLA-4
in mammals in need of such treatment which comprises administering
to said mammal an effective amount of a pharmaceutical composition
as described herein. In a preferred embodiment the mammal in need
of treatment is suffering from multiple sclerosis or rheumatoid
arthritis. In another preferred embodiment the mammal in need of
treatment may be suffering from asthma or psoriasis.
According to a further aspect of the present invention there is
provided a method for inhibiting the interaction between MAdCAM-1
and the integrin .alpha.4.beta.7 in mammals in need of such
treatment which comprises administering to said mammal an effective
amount of a pharmaceutical composition as described herein or a
pharmaceutically acceptable salt thereof. In a preferred embodiment
the mammal in need of treatment is suffering from inflammatory
bowel disease and insulin-dependent diabetes.
According to another aspect of the present invention there is
provided the use of a cyclic peptide of the invention or a
pharmaceutically-acceptable salt thereof in the production of a
medicament for use in the treatment of a disease or medical
condition mediated by the interaction between VCAM-1 or fibronectin
and the integrin receptor VLA-4.
According to another aspect of the present invention there is
provided the use of a cyclic peptide of the invention or a
pharmaceutically-acceptable salt thereof in the production of a
medicament for use in the treatment of a disease or medical
condition mediated by the interaction between MAdCAM-1 and the
integrin .alpha.4.beta.7.
Synthetic Details
A cyclic peptide of the invention may be prepared by any process
well known in the art of peptide chemistry to be applicable to the
synthesis of analogous compounds. Thus, for example, a cyclic
peptide of the invention may be obtained by procedures analogous to
those disclosed in "Solid Phase Peptide Synthesis: A practical
approach" by Atherton and Sheppard (published by IRL press at
Oxford University Press, 1989). "Solid Phase Peptide Synthesis" by
Stewart and Young (published by the Pierce Chemical Company,
Illinois, 1984), "Principles of Peptide Synthesis" by M. Bodanszky
(published by Springer-Verlag, Berlin Heidelberg, 1984), "The
Practice of Peptide Synthesis" by M. Bodanszky and A. Bodanszky
(published by Springer-Verlag, Berlin Heidelberg, 1984), and a
series of books "Amino Acids, Peptides and Proteins" (volumes 1-26;
volume 26 published in 1995) (published by the Royal Society of
Chemistry, Cambridge, UK). In addition to books, a number of
reviews [e.g. "Solid Phase Peptide Synthesis: a Silver Anniversary
Report", G. Barany, N. Kneib-Cordonier and D. G. Mullen,
International Journal of Peptide and Protein Research, 30 (1987)
705-739; "Solid Phase Peptide Synthesis Utilising
9-Fluorenylmethoxycarbonyl Amino Acids", G. B. Fields and R. L
Noble, International Journal of Peptide and Protein Research, 35
(1990) 161-214] have also been published on the synthesis of
peptides. Synthetic advances are also published in the proceedings
of the American, European and Japanese Peptide Symposiums.
Synthesis may be achieved by automated or manual means.
According to another aspect of the present invention there is
provided a process for the manufacture of a cyclic peptide of the
invention selected from:
(a) assembling the required linear peptide in a stepwise manner
(adding one amino acid at a time) followed by selective removal of
any N- and C-terminal protecting groups, cyclisation and finally
deprotonation to give a cyclic peptide according to the invention
and, optionally, if desired, converting the product thus obtained
into a salt thereof;
(b) forming of an amide bond by coupling two peptide units, one
containing a carboxylic acid group, or a reactive derivative
thereof, and the other containing an amino group, such that a
protected or unprotected cyclic peptide of the invention is
produced, and if necessary, removing the protecting groups using
process (c) below and, optionally, if desired, converting the
product thus obtained into a salt thereof; and
(c) removing one or more conventional peptide protecting groups
from a protected cyclic peptide having a protecting group on the
acid group in the side chain of Asp to give a cyclic peptide of the
invention and optionally, simultaneously or subsequently, also
removing any additional conventional peptide protecting groups
present and, optionally, if desired, converting the product thus
obtained into a salt thereof.
The above deprotection and coupling steps can be performed either
on a solid support (Solid Phase Peptide Synthesis) or in solution
using normal techniques used in the synthesis of organic compounds.
With the exception of the solid support, all the other protecting
groups, coupling reagents, deblocking reagents and purification
techniques are similar in both the solid phase and solution phase
peptide synthesis techniques.
For the synthesis of peptides on the solid support, a suitable
resin is selected which can either provide a free carboxyl group
after cleavage from the resin or a peptide derivative which can be
selectively deprotected to give a C-terminal carboxyl group. The
solid support may consist of polystyrene beads,
polydimethylacrylamide beads, polydimethylacrylamide-polystyrene
composite (Polyhipe) or polystyrene-polyoxyethylene resin (Tentagel
resins). A few examples of suitable linker group containing solid
supports used in the solid phase synthesis of peptides are shown
below. In addition to the linkers shown, some other linkers such as
hydroxycrotonoylamidomethyl (HYCRAM) can also be used. The first
amino acid is then coupled to the resin by the methods described in
this application for the synthesis of peptides or by using any of
the coupling reagents used in the synthesis of peptides. Examples
of some of the coupling reagents are also described in this
application. ##STR11##
During the assembly of peptides, the amino acid functional groups
not participating in the reaction are protected by various
protecting groups. For example, the N-terminal and the side chain
amino groups can be protected by using 9-fluorenylmethoxycarbonyl
(Fmoc), t-butoxycarbonyl (Boc), biphenylisopropoxycarbonyl (Bpoc),
2-[3,5-dimethoxyphenyl]propyl-2-oxycarbonyl (Ddz),
adamantyloxycarbonyl (Adoc), allyloxycarbonyl (Aloc),
2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl and various
substituted benzyloxycarbonyl groups. These protecting groups can
be cleaved when required by the standard techniques (e.g. acid or
base treatment, catalytic hydrogenolysis and Pd(0) treatment or
zinc/acetic acid treatment).
Suitable protecting groups used for the protection of the
.alpha.-carboxyl or the side chain carboxyl groups include various
esters (e.g. methyl, ethyl, t-butyl, benzyl, nitrobenzyl, allyl and
9-fluorenylmethyl).
Suitable protecting groups used for the protection of the side
chain guanidino group in the peptides containing an arginine
residue include a nitro, adamantyloxycarbonyl,
4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr),
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulphonyl (Pbf) and
2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc) groups. Suitable
protecting groups used for the protection of the side chain
imidazole group in the peptides containing a histidine residue
include a trityl, tosyl, dinitrophenyl, Adoc, Boc or Fmoc
group.
The protecting group cleavage reactions can be performed at
temperatures between 4.degree. C. to 40.degree. C. (preferably at
room temperature, about 25.degree. C.). The cleavage reactions can
take between 10 minutes to 24 hours.
Suitable coupling methods used for the coupling of the individual
amino acids or the peptide fragments include the commonly used
azide, symmetrical anhydride, mixed anhydride and various active
esters and carbodiimides. In case of various carbodiimides (e.g.
dicyclohexyl- or diisopropyl-carbodiimides), a number of additives
[e.g. 1 -hydroxybenzotriazole and N-hydroxysuccinimide) may also be
added. In addition, the amino acid or fragment couplings can also
be achieved by using a number of other reagents, e.g.
1H-benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium
hexafluorophosphate (PyBOP), (2-(1H-benzotriazole-1
-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and (2-(1
H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU)].
The coupling reactions can be performed at temperatures between
-20.degree. C. to 40.degree. C. The time required for completion of
the reaction may be between 10 minutes to 24 hours.
Suitable purification methods for the intermediates and final
products include counter current distribution, ion exchange, gel
filtration and various other chromatographic techniques including
high pressure liquid chromatography (HPLC) along with many other
standard techniques used in organic chemistry (e.g. solvent
extraction and crystallisation).
The invention will now be illustrated by the following non-limiting
examples in which:
FIG. 1 illustrates the synthesis of compound no. 1 (Table 2);
FIG. 2 illustrates the synthesis of compound no. 20 (Table 2):
FIG. 3 illustrates synthesis of compound no. 24 (Table 2);
FIG. 4 illustrates the structure of D-Orn(CHMe.sub.2);
FIG. 5 illustrates the structure of D-Lys(CHMe.sub.2);
FIG. 6 illustrates the structure of D-Orn(Me.sub.2);
FIG. 7 illustrates the structure of D-hArg(Et).sub.2 ; and
FIG. 8 illustrates the structure of D-Arg(Pmc).
In FIGS. 4 to 8 arrowed bonds indicate attachment points or direct
bonds (i.e. not a --CH.sub.2 --group). For example, referring to
D-Lys(CHMe.sub.2) (FIG. 5) in compound 4 (Table 1), the arrowed
bond on the nitrogen will be attached to the --C(O)--at the
C-terminus of Val and the arrowed bond on the --C(O)--will be
attached to the nitrogen atom at the N-terminus of D-Ala.
The following abbreviations have been used:
hArg(Et).sub.2 homo-Arg(Et).sub.2 Orn ornithine.
EXAMPLES
Syntheses of Compounds 1-38 (See Tables 1 and 2)
The cyclic peptides according to the invention are numbers 1 to 38
in tables 1 and 2. They were obtained by cyclisation of the
corresponding precursor (generally linear) peptides, numbered 39 to
75 in table 1. Synthetic details for compound nos. 1, 20 and 24 are
described below in detail (see FIGS. 1 to 3). In the case of other
compounds, only the variations from the standard procedure are
mentioned.
Example 1
Synthesis of Compound 1 (FIG. 1)
##STR12##
1. The cyclic peptide (FIG. 1, step 5) was prepared by the solid
phase procedure using 2-chlorotritylchloride resin. After
assembling the partially protected linear peptide on the resin, the
peptide was cleaved from the resin and used in the subsequent steps
without any purification. However, the final product was purified
extensively by reverse phase high pressure liquid chromatography
(HPLC) before characterisation.
1.1. Synthesis of Compound 1 (Step 1, FIG. 1)
t-Butyl bromoacetate (4.88 g, 25 mmole) in dichloromethane (50 ml)
was added to a solution of t-butyl-1-piperazine carboxylate (4.65
g, 25 mmole) and triethylamine (3.5 ml, 25 mmole) in
dichloromethane (30 ml). The reaction mixture was stirred
overnight, filtered to remove the solids separated overnight and
the filtrate evaporated to dryness. The residue was partitioned
between ethyl acetate and water, the organic layer was then washed
with water, dried over MgSO.sub.4 and evaporated to dryness. The
residue was crystallised from ether-isohexane to yield the product
(5.66 g, 75%, m.p. 99-100.degree. C.). [Elemental analysis: Found C
59.8%, H 9,6%, N 9.1%; C.sub.15 H.sub.28 N.sub.2 O.sub.4 requires C
60.0%, H 9.4%, N 9.33%]. [Thin layer chromatography on silica gel
plates showed a single spot; Rf 0.38 in ethyl acetate-isohexane
(1:1) and 0.68 in methanol-chloroform (1:9)].
1.2. Synthesis of Compound 1 (Step 2, FIG. 1)
The compound described in section 1.1 (5 g, 16.6 mmole) was treated
with a mixture of trifluoroacetic acid-water (95:5; 50 ml) for 1
hour. The acid was removed by evaporation in vacuum and the
residual oil was triturated with ether to give a solid which was
collected, washed with ether and dried over P.sub.2 O.sub.5 /KOH
under vacuum (6.25 g, m.p. 177-182.degree. C.). The solid was then
dissolved in a mixture of water and acetone (1:1, 150 ml)
containing potassium carbonate (6.92 g, 3 equivalents).
9-Fluorenylmethyl-N-hydroxysuccinimide (5.66 g, 16.7 mmole) in
acetone (30 ml) was added over a period of 20 minutes with
stirring. The pH of the solution was maintained at about 9 by the
addition of M K.sub.2 CO.sub.3 solution. After stirring overnight
at room temperature, the acetone was removed by evaporation under
vacuum and the aqueous solution was acidified with KHSO.sub.4
solution. The product was extracted into ethyl acetate and the
solution was washed with water (6 times) and with saturated NaCl
solution. The organic layer was dried over MgSO.sub.4 and
evaporated to give an oil which solidified on trituration with
isohexane and ether (yield 3.72 g, 60%). A sample was
recrystallised from ethanol-ether, m.p. 179-182.degree. C.,
(M+H).sup.+ 367.
1.3. Synthesis of Compound 1 (Steps 3 and 4, FIG. 1)
The above Fmoc-piperazine derivative (732 mg, 2 mmole) in
dichloromethane (15 ml) and diisopropylethylamine (1.05 ml, 3
equivalents) were added to 2-chlorotritylchloride resin
Novabiochem., 2.05 g) and the reaction mixture was shaken gently
for 60 minutes. A 10% solution of diisopropylethylamine in methanol
(10 ml) was added and the shaking was continued for 10 minutes. The
resin was filtered off, washed successively with dichloromethane,
dimethylformamide, dichloromethane, ether and dried at 50.degree.
C. in a vacuum oven (weight 2.55 g).
The above resin was placed in a reaction vessel fitted with a
sintered glass disc. The following series of reactions were then
carried out manually to obtain the desired peptide resin. (a)
Removal of the Fmoc group with two treatments (1.times.5 minutes
and 1.times.15 minutes) of 20% piperidine in dimethylformamide
followed by five washes with dimethylformamide to remove excess
reagents and cleavage products. (b) Acylation with Fmoc-Val (1.70
g, 5 mmole) activated with O-(benzotriazol-1-yl)-1,1,3,3
tetramethyluronium hexafluorophosphate (HBTU)(1.90 g, 5 mmole) and
diisopropylethylamine (1.75 ml, 10 mmole) in dimethylformamide (8
ml) for 1 hour. The resin was again washed five times with
dimethylformamide to remove excess reagents.
The above deprotection and coupling cycles were repeated using
Fmoc-Asp(OBu.sup.t) (2.06 g, 5 mmole), Fmoc-Leu (1.77 g, 5 mmole),
Fmoc-MeIle (1.83 g, 5 mmole), Fmoc-D-Arg(Pbf) (3.76 g, 5 mmole). In
the case of Fmoc-D-Arg(Pbf), the amino acid derivative was
activated by O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) (1.90 g, 5 mmole). One half of the resin
was then deblocked and reacted with Fmoc-NH(CH.sub.2).sub.2 -COOH
(911 mg, 3 mmole) using HBTU (1.14 g, 3 mmole) and
diisopropylethylamine (1.05 ml, 6 mmole) to give the protected
pentapeptide derivative attached to the chlorotrityl resin (step
3). The N-terminal Fmoc group was cleaved with 20% piperidine in
dimethylformamide (1.times.5 minutes and 1.times.15 minutes) and
the peptide resin was washed successively with dimethylformamide,
dichloromethane and ether and dried in a vacuum oven at 50.degree.
C.
The peptide resin was treated with a mixture of acetic
acid-trifluoroethanol-dichloromethane (2:2:6) (25 ml) for 1 hour.
The resin was removed by filtration, and treated again with the
cleavage reagent for one hour. The combined filtrates were
evaporated and the residue triturated with ether to give the linear
pentapeptide derivative as an acetate salt (824 mg, 0.682 mmole).
The acetate salt was then converted to a hydrochloride salt by
dissolving it in a mixture of water-acetonitrile (2:1, 60 ml),
cooling to 0.degree. C., adding 1.05 equivalents of IN HCl and
freeze drying the contents.
1.4. Synthesis of Compound 1 (Step 5, FIG. 1)
The linear peptide was cyclised and deprotected by the procedures
described below for c(MeIle-Leu-Asp-Val-D-Ala-D-Ala-D-Arg)
(compound 24) in the equivalent steps to give the final cyclic
peptide end product (1).
2. Synthesis of c(MePhe-Leu-Asp-Val-D-Arg-D-Arg) (Compound 20, FIG.
2)
The cyclic peptide was prepared by the solid phase procedure using
2-chlorotritylchloride resin. The synthetic details are described
below. After assembling the partially protected linear peptide on
the resin, the peptide was cleaved from the resin and used in the
subsequent steps without any purification. However, the final
product was purified extensively by reverse phase high pressure
liquid chromatography (HPLC) before characterisation.
2.1. Preparation of Fmoc-Val-chlorotrityl Resin (Step 1, FIG.
2)
2-Chlorotritylchloride resin (Alexis Corporation, 1.31 mmole Cl/g;
10 g) was swollen in dichloromethane (40 ml) (dried over molecular
sieve) for 5 minutes. A solution of Fmoc-Val (3.39 g, 10 mmole) and
diisopropylethylamine (5.6 ml, 32 mmole) in dichloromethane (20 ml)
was added and the suspension was shaken mechanically for 45
minutes. Methanol (9 ml) and diisopropylethylamine (1 ml) were
added and the shaking was continued for a further five minute
period. The resin was collected by filtration and washed
successively with dichloromethane, dimethylformamide and
dichloromethane and used immediately for the synthesis in the next
step (2.2)
2.2. Preparation of D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBut)-Va
-chlorotrityl Resin (Steps 2 and 3, FIG. 2)
The above Fmoc-Val resin was placed in a reaction vessel fitted
with a sintered glass disc. The following series of reactions were
then carried out manually to obtain the desired peptide resin.
(a) Removal of the Fmoc group with two treatments (1.times.5
minutes and 1.times.15 minutes) of 20% piperidine in
dimethylformamide followed by five washes with dimethylformamide to
remove excess reagents and cleavage products.
(b) Acylation with Fmoc-Asp(OBu.sup.t) (6.17 g, 15 mmole),
activated with O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) (5.70 g, 15 mmole) and
diisopropylethylamine (5.25 ml, 30 mmole) in dimethylformamide (22
ml) for 1 hour. The resin was again washed five times with
dimethylformamide to remove excess reagents.
The above deprotection and coupling cycles were repeated using
Fmoc-Leu (5.29 g, 15 mmole), Fmoc-MePhe (6.01 g, 15 mmole),
Fmoc-D-Arg(Pbf) (11.27 g, 15 mmole) and Fmoc-D-Arg(Pbf) (11.27 g,
15 mmole) to give
Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBu.sup.t)-Val-chlorotrityl
resin. As in the case of compound 1, coupling of the Fmoc-D-Arg
derivative was achieved by using
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) and diisopropylethylamine. The
N-terminal Fmoc group was cleaved (step 3) with 20% piperidine in
dimethylformamide (1.times.5 minutes and 1.times.15 minutes) and
the peptide resin,
D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBut)-Val-chlorotrityl resin,
was washed successively with dimethylformamide and
dichloromethane.
23. Preparation of
D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBu.sup.t)-Val, HCl. (Step 4,
FIG. 2)
The peptide resin,
D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBu.sup.t)-Val-chlorotrityl
resin, was suspended in a mixture of acetic
acid-trifluoroethanol-dichloromethane (2:2:6) (100 ml) for 1 hour.
The resin was removed by filtration and retreated with the same
mixture for a further one hour. The combined filtrates were
evaporated and the residue triturated with ether to give
D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBu.sup.t)-Val as an acetate
salt (11.66 g). The acetate salt was then converted to a
hydrochloride salt by dissolving it in a mixture of
water-acetonitrile (2:1, 350 ml), cooling to 0.degree. C., adding
1.05 equivalents of 1N HCl and freeze drying the contents (weight
11.5 g).
2.4. Preparation of
c(D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBut)-Val) (Step 5, FIG.
2)
The above linear peptide hydrochloride,
D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBu.sup.t)-Val (HCl), (11.5 g,
8.1 mmole) was dissolved in dimethylformamide (8000 ml) and
O-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) (4.62 g, 12.15 mmole), and
diisopropylethylamine (5.67 ml, 32.4 mmole) were added to the
solution. The cyclisation reaction was monitored by analytical
HPLC. On completion of the reaction (2 hours at room temperature),
the reaction mixture was evaporated to dryness in vacuum. The
residue was triturated with 10% aqueous sodium bicarbonate
solution. The solid was collected and washed with 10% sodium
bicarbonate, water, 10% potassium hydrogen sulphate solution and
finally with water. The solid was dried over P.sub.2 O.sub.5 at
45.degree. C. in a vacuum oven [retention time 24.80 minutes on a
Vydac 218TP54 column using a gradient of acetonitrile-water
containing 0.1% trifluoroacetic acid (40-80%) over a period of 30
minutes at a flow rate of 1.0 ml/minute] and was used in the next
step without any purification.
2.5. Preparation of c(MePhe-Leu-Asp-Val-D-Arg-D-Arg) [Compound 20]
(Step 6, FIG. 2)
The above protected cyclic peptide,
c(D)-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBu.sup.t)-Val), was treated
for 90 minutes with a mixture of trifluoroacetic acid-water (95:5,
80 ml) and triisopropylsilane (3 ml) to remove the arginine and
aspartic acid side chain protecting groups. The reaction mixture
was evaporation to a small volume and partitioned between water and
ether. The aqueous layer was washed 4 times with ether and freeze
dried to give 10.9 g crude product. The crude product was purified
by preparative reverse phase HPLC on a Vydac C.sub.18 218TP1015100
column (4 inch.times.25 cm) using a gradient of acetonitrile-water
containing 0.1% trifluoroacetic acid (15-35%) over a period of 80
minutes at a flow rate of 180.0 ml/minute. The product-containing
fractions were combined and freeze dried to give the purified
cyclic peptide (3.63 g). The peptide was characterised by amino
acid analysis and mass spectroscopy (table 2).
3. Synthesis of c(D-Arg-MeIle-Leu-Asp-Val-D-Ala-D-Ala) (Compound
24, FIG. 3)
The cyclic peptide was prepared by the solid phase procedure using
2-chlorotritylchloride resin. After assembling the partially
protected linear peptide on the resin, the peptide was cleaved from
the resin and used in the subsequent steps without any
purification. However, the final product was purified extensively
by reverse phase high pressure liquid chromatography (HPLC) before
characterisation.
3.1. Preparation of Fmoc-D-Ala-chlorotrityl Resin (Step 1, FIG.
3)
2-Chlorotritylchloride resin (Nova Biochem.; 1.35 mmole Cl/g; 1 g)
was swollen in dichloromethane (10 ml) (dried over molecular sieve)
for 5 minutes. A solution of Fmoc-D-Ala (311 mg, 1 mmole) and
diisopropylethylamine (525 .mu.l, 3 mmole) in dichloromethane (8
ml) was added and the suspension was shaken mechanically for 45
minutes. Methanol (9 ml) and diisopropylethylamine (1 ml) were
added and the shaking was continued for a further five minute
period. The resin was collected by filtration and washed
successively with dichloromethane, dimethylformamide,
dichloromethane, isopropanol and ether, and finally dried at
50.degree. C. in a vacuum oven (weight 1.51 g)
3.2. Preparation of
D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val-D-Ala-D-Ala-chlorotrityl Resin
(Steps 2 and 3, FIG. 3)
The above Fmoc-D-Ala resin was placed in a reaction vessel fitted
with a sintered glass disc. The following series of reactions were
then carried out manually to obtain the desired peptide resin.
(a) Removal of the Fmoc group with two treatments (1.times.5
minutes and 1.times.15 minutes) of 20% piperidine in
dimethylformamide followed by five washes with dimethylformamide to
remove excess reagents and cleavage products.
(b) Acylation with Fmoc-D-Ala (622 mg, 2 mmole), activated with
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) (760 mg, 2 mmole) and
diisopropylethylamine (700 .mu.l, 4 mmole) in dimethylformamide (3
ml) for 1 hour. The resin was again washed five times with
dimethylformamide to remove excess reagents.
The above deprotection and coupling cycles were repeated using
Fmoc-Val (678 mg, 2 mmole), Fmoc-Asp(OBu.sup.t) (822 mg, 2 mmole),
Fmoc-Leu (700 mg, 2 mmole), Fmoc-MeIle (734 mg, 2 mmole) and
Fmoc-D-Arg(Pmc) (1.40 g, 2 mmole) to give
Fmoc-D-Arg(Pmc)-Melle-Leu-Asp(OBu.sup.t)-Val-D-Ala-D-Ala-chlorotrityl
resin. As in the case of compound 1, coupling of the Fmoc-D-Arg
derivative was achieved by using
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) and diisopropylethylamine. The
N-terminal Fmoc group was cleaved (step 3) with 20% piperidine in
dimethylformamide (1.times.5 minutes and 1.times.15 minutes) and
the peptide resin,
D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val-D-Ala-D-Ala-chlorotrityl resin,
was washed successively with dimethylformamide, dichloromethane and
ether and dried in a vacuum oven at 50.degree. C. (weight 2.5
g).
3.3. Preparation of
D-Arg(Pmc)-MeIle-Leu-Asp(OBu.sup.t)-Val-D-Ala-D-Ala (HCI) (Step 4,
FIG. 3)
The peptide resin,
D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val-D-Ala-D-Ala-chlorotrityl resin,
was suspended in a mixture of-acetic
acid-trifluoroethanol-dichloromethane (2:2:6) (25 ml) for 1 hour.
The resin was removed by filtration and retreated with the same
mixture for a further one hour. The combined filtrates were
evaporated and the residue triturated with ether to give
D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val-D-Ala-D-Ala as an acetate salt
(1.18 g). The acetate salt was then converted to a hydrochloride
salt by dissolving it in a mixture of water-acetonitrile (2:1, 60
ml), cooling to 0.degree. C., adding 1.05 equivalents of 1N HCl and
freeze drying the contents.
3.4. Preparation of
c(D-Arg(Pmc)-MeIle-Leu-Asp(OBu.sup.t)-Val-D-Ala-D-Ala) (Step 5,
FIG. 3)
The above linear peptide hydrochloride,
D-Arg(Pmc)-MeIle-eu-Asp(OBu.sup.t)-Val-D-Ala-D-Ala (HCl), (1.18 g,
1.02 mmole) was dissolved in dimethylformamide (1000 ml) and
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) (395 mg, 1.02 mmole), and
diisopropylethylamine (545 .mu.l, 3.1 mmole) were added to the
solution. The cyclisation reaction was monitored by analytical
HPLC. On completion of the reaction (2 hours at room temperature),
the reaction mixture was evaporated to dryness in vacuum. The
residue was partitioned between ethyl acetate and water. The
organic layer was then washed successively with 1M citric acid,
saturated sodium chloride, 10% sodium bicarbonate, and saturated
sodium chloride, dried over magnesium sulphate and evaporated to
dryness in vacuum. The product was collected [retention time 27.06
minutes on a Vydac 218TP54 column using a gradient of
acetonitrile-water containing 0.1% trifluoroacetic acid (20-80%)
over a period of 30 minutes at a flow rate of 1.0 ml/minute] and
was used in the next step without any purification.
3.5. Preparation of c(D-Ala-D-Ala-D-Arg-MeIle-Leu-Asp-Val)
[Compound 24] (Step 6, FIG. 3)
The above protected cyclic peptide,
c(D-Ala-D-Ala-D-Arg(Pmc)-MeIle-Leu-Asp(OBu.sup.t)-Val), was treated
for 4 hours with a mixture of trifluoroacetic acid-water (95:5, 30
ml) and triisopropylsilane (1 ml) to remove the arginine and
aspartic acid side chain protecting groups. Evaporation to a small
volume, followed by trituration with ether yielded the crude cyclic
peptide (575 mg). The crude product was purified by preparative
reverse phase HPLC on a Deltapak C.sub.18 column (30.times.30 mm)
using a gradient of acetonitrfle-water containing 0.1I%
trifluoroacetic acid (10-30%/) over a period of 80 minutes at a
flow rate of 30.0 ml/minute. The product-containing fractions were
combined and freeze dried to give the purified cyclic peptide (353
mg). The peptide [single peak on HPLC, retention time 21.19 minutes
on a Vydac 218TP54 column using a gradient of acetonitrile-water
containing 0.1% trifluoroacetic acid (10 -40%) over a period of 30
minutes at a flow rate of 1.0 ml/minute] was characterised by amino
acid analysis and mass spectroscopy (table 2).
4. Synthesis of Compound 2
Compound 2 was synthesised by the route used for compound 1 (shown
in FIG. 1) except that .gamma.-aminobutyric acid was used in place
of .beta.-alanine.
5. Syntheses of Compounds 3 to 5
The above cyclic peptides were prepared by reacting the cyclic
peptide c(MeIle-Leu-Asp-Val-D-Orn-D-Orm) or
c(MeIle-Leu-Asp-Val-D-Lys-D-Ala) with the required aldehyde or
ketone and sodium cyanoboreohydride. For example, compound 3 was
prepared by dissolving the cyclic peptide
c(MeIle-Leu-Asp-Val-D-Orn-D-Orn) (100 .mu.mole) in dry acetone (2
ml) and reacting with sodium cyanoborohydride (IO equivalents).
After an hour, the reaction mixture was evaporated to dryness and
the residue, dissolved in water (5 ml), was acidified with acetic
acid and evaporated under high vacuum. The crude peptide was
purified by HPLC.
The parent cyclic peptides c(MeIle-Leu-Asp-Val-D-Orn-D-Orn) and
c(MeIle-Leu-Asp-Val-D-Lys-D-Ala) were synthesised by the route
described for compound 24 in FIG. 3.
6. Syntheses of Compounds 6 to 8
The linear peptides, D-hArg(Et).sub.2
-MeIle-Leu-Asp(OBut)-Val-D-Ala, D-hArg(Et).sub.2
-MeIle-Leu-Asp(OBut)-Val-D-Phe and D-hArg(Et).sub.2
-D-hArg(Et).sub.2 -Melle-Leu-Asp(OBut)-Val, required for compounds
6 to 8 were obtained by the procedures described for compound 24.
The synthesis was started from the required C-terminal amino acid,
i.e. D-Ala for compound 6, D-Phe for compound 7 and Val for
compound 8). The synthetic routes to Boc-D-hArg(Et).sub.2 have been
reported earlier in the literature [H.B. Arzeno et al., Synthetic
Communications, 20 (1990) 3433-3437; J. J. Nestor, Jr. et al.,
Journal of Medicinal Chemistry, 35 (1992) 3942-3948]. This
derivative was converted to Fmoc-D-hArg(Et).sub.2 by the standard
procedures and used in the syntheses of the above linear peptides.
Compounds 6 to 8 were then obtained from the corresponding linear
peptides by the cyclisation methods described for compound 24 in
FIG. 3.
7. Syntheses of Compounds 9 to 19
Cyclic peptides 9 to 19 were synthesised by the procedures similar
to that described for compound 24. As mentioned in table 1, all the
linear peptides were synthesised on the chlorotrityl resin starting
from Fmoc-Valine.
8. Syntheses of compounds 21 to 23 and 25 to 32
Cyclic peptides 21 to 23 and 25 to 32 were synthesised by the
procedures similar to that described for compound 20. As mentioned
in table 1, all the linear peptides were synthesised on the
chlorotrityl resin starting from Fmoc-Valine.
9. Syntheses of Compounds 33 to 35
Cyclic peptides 33 to 35 were synthesised by the procedures similar
to that described for compound 20. However, as shown in table 1,
the three linear peptides were synthesised on the chlorotrityl
resin starting from Fmoc-NH(CH.sub.2).sub.5 -COOH,
Fmoc-NH(CH.sub.2).sub.2 --S--CH.sub.2 --COOH or
Fmoc-NH(CH.sub.2).sub.2 --S--(CH.sub.2).sub.2 --COOH derivatives in
place of Fmoc-Valine.
Unlike aminohexanoic acid which is commercially available, the
other two sulphur containing amino acid derivatives were
synthesised by the following procedures. Fmoc-NH(CH.sub.2).sub.2
--S--CH.sub.2 --COOH (used above) was obtained from
2-aminoethanethiol and 2-bromoacetic acid. 2-Aminoethanethiol
hydrochloride (5.68 g, 50 mmole) was dissolved in water (200 ml)
and sodium hydrogen carbonate (25.2 g, 300 mmole) was added to it.
2-Bromoacetic acid (6.95 g, 50 mmole) dissolved in acetonitrile
(100 ml) was added in portions over 30 minutes to the stirred
solution prepared above. After 1 hour at room temperature, a
solution of 9-fluorenylmethyl-N-hydroxysuccinimide (Fmoc-OSu)
(16.85 g. 50 mmole) in acetonitrile (150 ml) was added and the
stirring was continued for 16 hours. The slightly turbid solution
was evaporated to remove-most of-the acetonitrile and the remaining
aqueous solution was extracted with ethyl acetate (3.times.50 ml)
and acidified (pH 2) by the addition of hydrochloric acid. The
white solid wHs collected, washed with water and dried in vacuo at
45.degree. C. Yield 17 g (95%), (M+H).sup.+ 358.0.
Fmoc-NH(CH.sub.2).sub.2 --S--(CH.sub.2).sub.2 --COOH was obtained
by the procedure described above for Fmoc-NH(CH.sub.2).sub.2
--S--CH.sub.2 --COOH by using 3-bromopropionic acid and
2-aminoethanethiol. (M+H).sup.+ 372.
10. Syntheses of Compounds 36 to 38
Cyclic peptides 36 to 38 were synthesised by the procedures similar
to that described for compound 20. As mentioned in table 1, all the
linear peptides, except compound 61, were synthesised on the
chlorotrityl resin starting from Fmoc-Valine. As shown in FIG. 2,
compound 61 was prepared starting from Fmoc-Ala. The sulphur
containing amino acid residue, --NH(CH.sub.2).sub.2 --S--CH.sub.2
--CO--, was incorporated by using Fmoc-NH(CH.sub.2).sub.2
--S--CH.sub.2 --COOH at the appropriate stage in the synthesis of
the linear peptides.
Example 2
In Vitro and In Vivo Assays
The following abbreviations and sources of materials are used in
this example. MOLT-4 cells--lymphocytic T cell line (ATCC derived)
Fibronectin --Made according to the methods described in
E.Nengvall, E. Ruoslahti, Int. J. Cancer, 1977, 20, pages 1-5 and
J. Forsyth et al, Methods in Enzymology, 1992, 215, pages 311-316)
or reagent grade human fibronectin. The later was purified from
human plasma by gelatin-sepharose affinity chromatography. Source:
Bio Products Elstree UK. Product No. 9136. A review article on
fibronectins is Fibronectins--Adhesive Glycoproteins of Cell
Surface and Blood, K. M. Yamada and K. Olden, Nature, 275 (1978)
179-184. rsVCAM-1-- (Reference source: Biochem Biophys Res Comm
1991 178 N3; 1498-1504). VCAM-1 is a cell surface glycoprotein
produced by the vascular endothelium, as well as on macrophage-like
and dandritic cell types, in response to certain inflammatory
stimuli. VCAM-1 interacts with the integrin VLA-4 present on
mononuclear leukocytes. The cDNA for VCAM-1 was isolated by
screening a cDNA library from IL-1.beta.-activated human
endothelial cells. Large quantities of the Stein were expressed in
insect cells using a baculovirus expression system. VCAM-1
expressing cells were shown to bind specifically to a variety of
VLA-4 expressing cell lines (Jurkat, THP-1, U937). Another
reference on VCAM-1 is Expression and Functional Characterisation
of Recombinant Human Vascular Cell Adhesion Molecule-1 (VCAM-1)
Synthesised by Baculovirus-Infected Insect Cells, J. K.
Stoltenborg, R. A. Straney, R. J. Tritch, W. M. Mackin and H. J.
George, Protein Expression and Purification, 4 (1993) 585-593.
RPMI 1640--Cell media. Source Gibco BRL (Life technologies; Cat No
31870-025).
FCS--Foetal calf serum. Source Advanced protein products (West
Midlands UK) Cat No AS-302-50.
BCECF-AM--2', 7'-bis (2
carboxyethyl)-5-(.epsilon.6)-carboxyfluoroscein acetoxymethyl
ester). source: Molecular Probes Inc USA; Cat No B-1 150.
CHO DG44--Chinese hamster ovary cell line (ATCC derived; Reference:
Som Cell Mol Gen 1986; 12; 555-666)
DMEM--Dulbecco's modified eagle medium. Source Gibco BRL (Life
technologies; Cat No 41966-029.
Antibiotic--Penicillin-streptomycin. Source Gibco BRL (Life
Technologies; Cat No 15070-022).
Fluorskan.TM.--is a fluorimeter.
HUVEC--Human umbilical vein endothelial cells. Primary cultures
prepared from tissue samples. (Reference: J Clin Invest. 1973 52;
2745-2747. Recombinant human TNFA.alpha.--Tumor necrosis factor
Alzet osmotic minipump--Subcutaneous implanted micro osmotic pump,
Alza Corporation Palo Alto, Calif.
In the following assays and models references to compound(s) refers
to cyclic peptide(s) of the present invention.
2.1 Cell/Immobilised Ligand Assays
2.1.1 MOLT4 cell/Fibronectin-VCAM-1 Adhesion Assay.
The MOLT4 cell/Fibronectin-VCAM-1 adhesion assay was used to
investigate the interaction of the integrin VLA4 (Very Late
Antigen, .alpha.4/.beta.1) expressed on the MOLT-4 cell membrane
with fibronectin or recombinant soluble VCAM-1 (rsVCAM-1).
Fibronectin or rsVCAM-1 were coated overnight at 4.degree. C. onto
polystyrene 96-well microtitre plates at concentrations of
20.mu.g/ml and 1 .mu.g/ml respectively. Following this, a
concentrated BSA solution (10 mg/ml) was added to block
non-specific binding sites. After aspiration of these solutions,
equal volumes of compound and MOLT-4 cell suspension (1.times.10E6
cells/ml) were added. Adhesion took place during a 2 hour
incubation at 37.degree. C., non or loosely adherent cells were
removed by gentle agitation followed by vacuum aspiration.
Quantitation of the remaining adherent cells was by means of a
colorimetric assay of acid phosphatase activity, which was read on
a spectrophotometer. Compounds which inhibited adhesion gave a
lower absorbance reading. Standard, control and test conditions
were assayed in triplicate. Percentage inhibition was calculated
with respect to total (no inhibitor) and non-specific (no
fibronectin) standards on each plate.
2.1.2 JY cell/MAdCAM-1 Adhesion Assay
The JY cell (human B lymphoblastoid)/MAdCAM-1 adhesion assay was
used to investigate the interaction of the integrin .alpha.4.beta.7
expressed on the JY cell membrane with recombinant soluble
MAdCAM-1. MAdCAM-1 was coated onto polystyrene 96-well microtitre
plates for 1 hour at a concentration of 10 .mu.g/ml. Following
this, a concentrated BSA solution (10 mg/ml) was added to block
non-specific binding sites. After aspiration of these solutions,
equal volumes of compound and JY cell suspension (1.times.10E6
cells/ml) were added. The assay contained manganese at a final
concentration of 0.2 mM to activate the .alpha.4.beta.7 on the JY
cells. Adhesion took place during a 20 minute incubation at
37.degree. C. Non or loosely adherent cells were removed by gentle
agitation followed by vacuum aspiration. Adherent cells were then
fixed with 5% glutaraldehyde for 20 minutes and stained for 20
minutes with a 0.1% solution of crystal violet. The crystal violet
was solublised by adding 10% acetic acid and the number of adherent
cells quantitated by measuring absorbance at 405 nm on a
spectrophotometer. Compounds which inhibited adhesion resulted in a
lower absorbance reading. Standard, control and test conditions
were assayed in quintuplicate. Percentage inhibition was calculated
with respect to total (no inhibitor) and non-specific (no MAdCAM-1)
standards on each plate.
2.2 Cell-Cell Assays
2.2.1. VCAM-1 CHO cells
MOLT4 cells (RPMI 1640 supplemented with 5% FCS and 2mM
L-Glutamine) were labelled with the fluorescent dye BCECF-AM (30
.mu.g/ml per 3.times.10E6 cells). CHO DG44 transfected with full
length VCAM-1 cDNA were selected for VCAM-1 expression by FACS
analysis and grown to confluence in 96 well tissue culture plates.
Prior to use in the adhesion assay CHO DG44 cells were washed three
times (DMEM supplemented with 5% FCS, 2mM L-Glutamine and 2%
antibiotic). MOLT-4 (10E5 cell/well) cells were over laid on the
VCAM-1 expressing CHO cells and incubated for 30 minutes at
37.degree. C., 5% CO.sub.2. The non-adherent cells were removed by
washing the plate three times (RPMI 1640 supplemented with 5% FCS
and 2mM L-Glutamine) following which the plates were blotted dry on
tissue paper. 100 .mu.l of 2% Triton X-100 was added to each well
and the plates read using a Fluoroskan (excitation=485 nM,
emission=538 nM). Compounds were dissolved in appropriate solvents
and added to the MOLT-4 cells prior to addition to HUVEC cultures.
is Inhibition of adhesion was calculated by comparing level of
adhesion (fluorescence) of control vehicle treated cells with
compound treated cells.
2.2.2 Human Umbilical Vein Endothelial Cells.
MOLT4 cells (RPMI 1640 supplemented with 5% FCS and 2mM
L-Glutamine) were labelled with the fluorescent dye BCECF-AM (30
.mu.g/ml per 3.times.10E6 cells). Primary HUVEC was grown to
confluence in 96 well tissue culture plates and incubated for 18
hours with 2 U/ml recombinant human TNF.alpha.. Prior to use in the
adhesion assay the primary HUVEC monolayers were washed (M199
supplemented with 5% FCS, 2mM L-Glutamine and 2% antibiotic).
MOLT-4 (10E5cell/well) cells were overlaid on the primary HUVEC and
incubated for 30 minutes at 37.degree. C., 5% CO.sub.2. The
non-adherent cells were removed by washing the plate three times
(RPMI 1640 supplemented with 5% FCS and 2mM L-Glutamine) and dried
by blotting on tissue paper. 100 .mu.l of 2% Triton X-100 was added
to each well and the plates read using a Fluoroskan (excitation=485
nM, emission=538 nM). Compounds were dissolved in appropriate
solvents and added to the MOLT-4 cells prior to addition to HUVEC
cultures. Inhibition of adhesion was calculated comparing level of
adhesion (fluorescence) of control vehicle treated cells with
compound treated cells.
2.3 In Vivo Contact Hypersensitivity Response
Balb/C male mice (20-25g) are sensiti'sed with oxazolone (50 .mu.l
of 0.24% in acetonelolive oil) by topical application to the shaved
skin area of the back. Seven days later the mice are challenged by
topical application of oxazolone (25 .mu.l of 0.25% in
acetone/olive oil) to the surface of the ear. Swelling of the ear
develops over a 24 hour period following which ear thickness is
measured and compared to the pre-challenge thickness, the
percentage increase in ear thickness is calculated. Compounds are
delivered by continuous infusion at doses within the range from 10
mg/kg/day to 0.001 mg/kg/day from subcutaneous Alzet osmotic
minipumps which are implanted 24 hours prior to the oxazolone
challenge. Inhibition of the inflammatory response is calculated
comparing vehicle treated animals and compound treated groups (n=6
animals per group).
2.4 In Vivo Ovalbumin Delayed Type Hypersensitivity Model.
Balb/C female mice (20-25 g) were immunised on the flank with an
emulsion of ovalbumin (Sigma; 0.1 ml subcutaneous injection of 2
mg/ml solution mixed (1:1) with complete Freunds adjuvant; Difco).
Seven days later the mice were challenged by subplantar injection
of ovalbumin (30 .mu.l of 1% heat aggregated ovalbumin in saline)
into the left hind foot pad. Swelling of the foot developed over a
24 hour period following which foot pad thickness was measured and
compared to the pre-challenge thickness. The percentage increase in
foot pad thickness was calculated. Compounds were delivered by
continuous infusion at doses within the range from 10 mg/kg/day to
0.001 mg/kg/day from subcutaneous Alzet osmotic minipumps which
were implanted 24 hours prior to the ovalbumin challenge.
Inhibition of the inflammatory response was calculated comparing
vehicle treated animals and compound treated groups (n=5 animals
per group).
2.5 In Vivo Antigen Induced Arthritis Model.
Mice are inmnunised and boosted 7 days later with a combination of
100 .mu.g methylated BSA in complete Freund's adjuvant (s.c.)
followed by an intraperitoneal injection of bordetella pertussis
organisms. Two weeks after boost animals are challenged with 100
.mu.g methylated-bovine serum albumin (BSA) intra-articularly and
the degree of inflammation/arthritis determined by measuring knee
joint swelling, histology and changes in acute phase proteins.
Compounds are delivered by continuous infusion at a dose rate
ranging from 30 mg/kg/day to 0.001 mg/kg/day from subcutaneous
Alzet osmotic minipumps which are implanted 24 hours prior to the
challenge. The degree of inflammation/arthritis is compared with
the control animals and contralateral knee.
2.6 Experimental Autoimmune Encephalomyelitis Model.
Disease was induced by s.c. injection of a mixture of spinal cord
homogenate, myelin basic protein (MBP) or encephalogenic peptides
with complete Freund's adjuvant (CFA), coupled with an i.p.
injection of pertussis toxin. For acute disease, pertussis
injection was repeated 2 days after immunisation. For chronic
disease, pertussis was omitted and mice received two injections of
antigen in CFA, with an interval of 7 days. Disease was assessed by
clinical scoring supported by histology. Compounds were dosed by
continuous infusion at doses within the range from 10 mg/kg/day to
0.001 mg/kg/day from subcutaneous Alzet osmotic minipumps which
were implanted 24 hours prior to challenge. Symptoms were compared
with the control animals.
2.7 Mouse Bronchiolar Lavage Eosinophilia Model
Male C57BL/6J mice (20-25 g) were sensitised by i.p. injection of
0.1 ml saline containing 100 .mu.g ovalbumin and 2.5 mg aluminium
hydroxide three times at 4 to 5 day intervals. Ten to fourteen days
after the last sensitising injection the animals were placed in a
Perspex chamber and exposed (0.5 to 1 h) to aerosolised ovalbumin
dissolved in saline (up to 15 mg/ml) delivered from a DeVilbiss
Aerosonic nebuliser into a stream of air entering the chamber at
approximately 3 l/min. Mice were exposed to aerosolised ovalbumin
for 2 to 4 periods over 3 to 4 days and the day after the final
exposure were killed by overdose of sodium barbitone. The trachea
of each mouse was exposed, cannulated with a ball-tipped needle and
bronchioalveolar lavage performed with 4.times.1 ml saline. The
cells in the pooled lavage fluid were coated onto a glass
microscope slide and fixed and stained with Leukostat stain.
Differential cell counts were performed by counting a minimum of
200 leukocytes and the percentage of eosinophils was calculated.
Test compounds were delivered at doses within the range from 10
mg/kg/day to 0.001 mg/kg/day from subcutaneous Alzet osmotic
minipumps implanted 24 h before aerosolised ovalbumin challenge.
Inhibition of eosinophilia was calculated by comparing mice dosed
with vehicle or compound (n=5-10 per group).
2.8 Collagen-Induced Arthritis Model
DBA/1 male mice (ex Harlan/Olac U.K.)-were immunised with 0.1 ml of
a mixture of equal parts of bovine collagen type II in 0.05 M
acetic acid at 2 mg/ml and complete Freunds adjuvant (Sigma). This
mixture was injected at the base of the tail. Twenty days later
compounds or vehicles were delivered by continuous infusion at
doses within the range from 10 mg/kg/day to 3 mg/kg/day from
subcutaneous Alzet osmotic minipumps. On the following day, each
animal received an intra-peritoneal booster injection of 0.1 ml of
collagen type II in acetic acid. The degree of arthritis of vehicle
treated and compound treated animals was compared.
TABLE 1 Synthesis and purification of cyclic peptides. High
Pressure Liquid Chromatography (HPLC) NO: Precursor NO: End Product
Cyclic Peptide (Gradient system and time) 39 ##STR13## 1 ##STR14##
Deltapak column 15-30% (80 min.) 40 ##STR15## 2 ##STR16## Deltapak
column 10-30% (80 min.) 41 c(MeIle-Leu-Asp-Val-D-Orn-D-Orn) 3
c(MeIle-Leu-Asp-Val-D-Om(CHMe.sub.2)-D- 10-40% (70 min.)
Orn(CHMe.sub.2)) (12 ml/min.) 42 c(MeIle-Leu-Asp-Val-D-Lys-D-Ala) 4
c(MeIle-Leu-Asp-VaI-D-Lys(CHMe.sub.2)-D-Ala) Dynamax column 10-50%
(60 min.) 41 c(MeIle-Leu-Asp-Val-D-Orn-D-Orn) 5
c(MeIle-Leu-Asp-Val-D-Orn(Me.sub.2)-D-Orn(Me.sub.2)) 15-45% (65
min.) (15 ml/min.) 43 D-hArg(Et).sub.2
-MeIle-Leu-Asp(OBut)-Val-D-Ala 6
c(MeIle-Leu-Asp-Val-D-Ala-D-hArg(Et).sub.2) 10-50% (60 min.) 44
D-hArg(Et).sub.2 -MeIle-Leu-Asp(OBut)-Val-D-Phe 7
c(MeIle-Leu-Asp-Val-D-Phe-D-hArg(Et).sub.2) 10-50% (60 min.) 45
D-hArg(Et).sub.2 -D-hArg(Et).sub.2 -MeIle-Leu-Asp(OBut)- 8
c(MeIle-Leu-Asp-Val-D-hArg(Et).sub.2 -D-hArg(Et).sub.2) 10-50% (60
min.) Val 46 D-Lys(Boc)-D-His(Trt)-MeIle-Leu-Asp(OBut)-Val 9
c(MeIle-Leu-Asp-Val-D-Lys-D-His) 10-30% (60 min.) 47
D-Arg(Pmc)-D-Lys(Boc)-MeIle-Leu-Asp(OBut)-Val 10
c(MeIle-Leu-Asp-Val-D-Arg(Pmc)-D-Lys) 10-60% (60 min.) 48
D-Lys(Boc)-D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val 11
c(MeIle-Leu-Asp-Val-D-Lys-D-Arg) 10-30% (60 min.) 49
D-Ala-D-Lys(Boc)-MePhe-Leu-Asp(OBut)-Val 12
c(MePhe-Leu-Asp-Val-D-Ala-D-Lys) 10-40% (60 min.) 50
D-Arg(Pmc)-D-Lys(Boc)-MeIle-Leu-Asp(OBut)-Val 13
c(MeIle-Leu-Asp-Val-D-Arg-D-Lys) 10-30% (60 min.) 51
D-Ala-D-Ala-Lys(Boc)-Leu-Asp(OBut)-Val 14
c(Lys-Leu-Asp-Val-D-Ala-D-Ala) 10-20% (60 min.) 52
D-Ala-D-Ala-Arg(Pmc)-Leu-Asp(OBut)-Val 15
c(Arg-Leu-Asp-Val-D-Ala-D-Ala) 5-20% (60 min.) 53
D-Ala-D-Lys(Boc)-D-Phe-Leu-Asp(OBut)-Val 16
c(D-Phe-Leu-Asp-Val-D-Ala-D-Lys) 10-30% (60 min.) 54
D-Ala-D-Ala-MePhe-Leu-Asp(OBut)-Val 17
c(Mephe-Leu-Asp-Val-D-Ala-D-Ala) 10-50% (60 min.) 55
D-Ala-D-Ala-D-Phe-Leu-Asp(OBut)-Val 18
c(D-Phe-Leu-Asp-Val-D-Ala-D-Ala) 20-40% (60 min.) 56
D-Ala-D-Lys(Boc)-D-Met-Leu-Asp(OBut)-Val 19
c(D-Met-Leu-Asp-Val-D-Ala-D-Lys) 5-20% (60 min.) 57
D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu-Asp(OBut)-Val 20
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg) 15-30% (100 min.) 58
D-Arg(Pbf)-D-His(Trt)-MePhe-Leu-Asp(OBut)-Val 21
c(MePhe-Leu-Asp-Val-D-Arg-D-His) 15-30% (100 min.) 59
D-Trp-D-Arg(Pbf)-MePhe-Leu-Asp(OBut)-Val 22
c(MePhe-Leu-Asp-Val-D-Trp-D-Arg) 25-35% (90 min.) 60
D-Arg(Pmc)-D-Ala-D-Arg(Pmc)-MePhe-Leu- 23
c(MePhe-Leu-Asp-Val-D-Arg-D-Ala-D-Arg) 5-30% (60 min.)
Asp(OBut)-Val 61 D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val-D-Ala-D 24
c(MeIle-Leu-Asp-Val-D-Ala-D-Ala-D-Arg) Deltapak column Ala 10-30%
(80 min.) 62 D-Arg(Pmc)-D-Ala-D-Arg(Pmc)-MeIle-Leu 25
c(MeIle-Leu-Asp-Val-D-Arg-D-Ala-D-Arg) 10-30% (60 min.)
Asp(OBut)-Val 63 D-Ala-D-Arg(Pmc)-D-Arg(Pmc)-MeIle-Leu 26
c(MeIle-Leu-Asp-Val-D-Ala-D-Arg-D-Arg) 10-30% (60 min.)
Asp(OBut)-Val 64 D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-MePhe-Leu- 27
c(MePhe-Leu-Asp-Val-D-Ala-D-Arg-D-Arg) 10-35% (60 min.)
Asp(OBut)-Val 65 D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-MeIle-Leu- 28
c(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Ala) 10-30% (60 min.)
Asp(OBut)-Val 66 D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-MePhe-Leu- 29
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg-D-Ala) 10-35% (60 min.)
Asp(OBut)-Val 67 D-Ala-D-Ala-D-Arg(Pmc)-D-Arg(Pmc)-MeIle-Leu- 30
c(MeIle-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg) 10-25% (60 min.)
Asp(OBut)-Val 68 D-Ala-D-Ala-D-Arg(Pmc)-D-Arg(Pmc)-MePhe-Leu- 31
c(MePhe-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D- 10-40% (60 min.)
Asp(OBut)-Val Arg) 69 Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Ala-D-MeIle-Leu-
32 c(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Ala-D-Ala) 4" Vydac column
Asp(OBut)-Val 15-35% (100 min.) 70
D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val- 33
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.5 CO) Deltapak column
NH(CH.sub.2).sub.5 COOH 15-30% (80 min.) 71
D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val-NH(CH.sub.2).sub.2 -- 34
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.2 --S--(CH.sub.2).sub.2
-- Deltapak column S--(CH.sub.2).sub.2 -COOH CO) 15-30% (80 min.)
72 D-Arg(Pmc)-MeIle-Leu-Asp(OBut)-Val-NH(CH.sub.2).sub.2 -- 35
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.2 --S--CH.sub.2 --
Deltapak column S--CH.sub.2 --COOH CO) 10-30% (80 min.) 73
D-Arg(Pmc)-NH(CH.sub.2).sub.2 --S--CH.sub.2 CO-MeIle-Leu- 36
c(MeIle-Leu-Asp-Val-D-Arg-NH(CH.sub.2).sub.2 --S--CH.sub.2 CO)
10-40% (60 min.) Asp(OBut)-Val 74 NH.sub.2 --CH.sub.2 --CH.sub.2
--S--CH.sub.2 --CO-D-Arg(Pmc)-D-Arg(Pmc)- 37
c(MeIle-Leu-Asp-Val-NH--CH.sub.2 --CH.sub.2 --S--CH.sub.2 --CO-D-
10-40% (60 min.) MeIle-Leu-Asp(OBut)-Val Arg-D-Arg) 75 NH.sub.2
--CH.sub.2 --CH.sub.2 --S--CH.sub.2 --CO-D-Arg(Pmc)-D-Arg(Pmc)- 38
c(MePhe-Leu-Asp-Val-NH--CH.sub.2 --CH.sub.2 --S--CH.sub.2 --CO-D-
10-30% (60 min.) MePhe-Leu-Asp(OBut)-Val Arg-D-Arg) Preparative
HPLC was carried out using a reverse phase (C.sub.18) 1 inch
diameter Vydac column (218TP1022, 22 .times. 250 mm). In some cases
(mentioned in the table) either a Deltapak column (30 .times. 300
mm) or a Vydac 4" column was used. The solvent system consisted of
water and acetonitrile (each containing 0.1% trifluoroacetic acid).
The column was eluted using a gradient (solvent ratio and time
shown in the table) with increasing concentrations of acetonitrile
run at a rate #of 10 ml/minute for the Vydac column, 30 ml/min. for
the Deltapak column and 100 ml/min. for the 4" Vydac column.
TABLE 2 Synthesis and Characterisation of the Cyclic Peptides Amino
Acid Analysis (Acid hydrolysis - 6N HCl HPLC Mass Comp. containing
1% phenol, Retention Time Spectroscopy No. End Product Cyclic
Peptide 24 hours, 130.degree. C.) (Min.) (M + H).sup.+ 1 ##STR17##
Asp 1.0, Val 0.96, Leu 1.04, .beta.-Ala 0.96, Arg 1.04 18.91 10-40%
(30 min.) 808.5 2 ##STR18## Asp 1.02, Val 0.96, Leu 1.04,
.gamma.-Abu 0.95, Arg 0.99 19.41 10-40% (30 min.) 822.5 3
c(MeIle-Leu-Asp-Val-D-Orn(CHMe.sub.2)-D- Asp 0.99, Val 0.97, Leu
1.04, Orn 2.01. 23.45 767.5 Orn(CHMe.sub.2)) 10-40% (30 min.) 4
c(MeIle-Leu-Asp-Val-D-Lys(CHMe.sub.2)-D-Ala) Asp 1.0, Ala 1.0, Val
0.96, Leu 0.97. 13.21 696.5 20-80% (40 min.) 5
c(MeIle-Leu-Asp-Val-D-Orn(Me.sub.2)-D-Orn(Me.sub.2)) Asp 0.96, Val
1.02, Leu 1.02. 25.82 740 10-60% (30 min.) 6
c(MeIle-Leu-Asp-Val-D-Ala-D-hArg(Et).sub.2) Asp 1.0, Ala 1.06, Val
0.95, Leu 0.99, 13.34 752.4 hArg(Et).sub.2, 0.97. 20-80% (40 min.)
7 c(MeIle-Leu-Asp-Val-D-Phe-D-hArg(Et).sub.2) Asp 1.0, Phe 0.96,
Val 0.95, Leu 0.99, 16.49 828.9 hArg(Et).sub.2, 0.98. 20-80% (40
min.) 8 c(MeIle-Leu-Asp-Val-D-hArg(Et).sub.2 -D-hArg(Et).sub.2) Asp
1.0, Val 0.97, Leu 0.98, 12.03 907.6 hArg(Et).sub.2, 2.1. 20-80%
(40 min.) 9 c(MeIle-Leu-Asp-Val-D-Lys-D-His) Asp 1.02, Val 1.0, Leu
1.02, Lys 0.95, 25.97 720.5 His 0.97. 10-30% (40 min.) 10
c(MeIle-Leu-Asp-Val-D-Arg(Pmc)-D-Lys) Asp 1.05, Val 1.0, Leu 0.99,
Lys 0.99, 1005.5 Arg 0.95. 11 c(MeIle-Leu-Asp-Val-D-Lys-D-Arg) Asp
1.05, Val 1.0, Leu 1.01, Lys 0.95, 8.98 739.5 Arg 0.99. 20-80% (40
min.) 12 c(MePhe-Leu-Asp-Val-D-Ala-D-Lys) Asp 0.96, Ala 1.0, Val
0.95, Leu 1.05, 24.27 688.2 Lys 0.95. 10-40% (40 min.) 13
c(MeIle-Leu-Asp-Val-D-Arg-D-Lys) Asp 1.05, Val 1.0, Leu 1.01, Lys
1.01, 19.37 739.5 Arg 0.96. 10-40% (40 min.) 14
c(Lys-Leu-Asp-Val-D-Ala-D-Ala) Asp 1.0, Ala 2.09, Val 0.96, Leu
0.96, 9.11 598.3 Lys 0.97. 10-40% (40 min.) 15
c(Arg-Leu-Asp-Val-D-Ala-D-Ala) Asp 1.0, Ala 2.06, Val 0.97, Leu
0.95, 11.84 626.4 Arg 0.95. 10-30% (40 min.) 16
c(D-Phe-Leu-Asp-Val-D-Ala-D-Lys) Asp 1.0, Ala 1.05, Val 0.97, Leu
0.97, 18.58 674.5 Lys 0.96, Phe 0.99. 10-40% (40 min.) 17
c(MePhe-Leu-Asp-Val-D-Ala-D-Ala) Asp 1.0, Ala 2.09, Val 0.97, Leu
0.98. 16.92 631.4 20-80% (40 min.) 18
c(D-Phe-Leu-Asp-Val-D-Ala-D-Ala) Asp 1.05, Ala 1.95, Val 1.0, Leu
0.98, 12.34 (M - H).sup.- Phe 0.95. 20-80% (40 min.) 615.3 19
c(D-Met-Leu-Asp-Val-D-Ala-D-Lys) Asp 1.05, Ala 1.0, Val 1.01, Leu
1.03, 13.23 658.3 Lys 1.03, Met 0.98. 10-40% (40 min.) 20
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg) Asp 1.00, Val 0.96, Leu 1.0, Arg
1.96. 21.19 801.4 10-40% (30 min.) 21
c(MePhe-Leu-Asp-Val-D-Arg-D-His) Asp 1.03, Val 1.01, Leu 1.0, Arg
1.0, 10.59 782.3 His 0.99. 20-35% (15 min.) 22
c(MePhe-Leu-Asp-Val-D-Trp-D-Arg) Asp 0.99, Val 0.97, Leu 1.0, Arg
1.03, 7.89 831.0 Trp 0.59. 30-45% (15 min.) 23
c(MePhe-Leu-Asp-Val-D-Arg-D-Ala-D-Arg) Asp 1.04, Ala 1.05, Val 1.0,
Leu 0.98, 20.24 872.6 Arg 1.99. 10-40% (40 min.) 24
c(MeIle-Leu-Asp-Val-D-Ala-D-Ala-D-Arg) Asp 0.98, Ala 2.03, Val
0.97, Leu 1.0, 21.19 753.4 Arg 1.01 10-40% (30 min.) 25
c(MeIle-Leu-Asp-Val-D-Arg-D-Ala-D-Arg) Asp 1.04, Ala 0.98, Val 1.0,
Leu 1.0, 18.41 838.4 Arg 2.01 10-40% (40 min.) 26
c(MeIle-Leu-Asp-Val-D-Ala-D-Arg-D-Arg) Asp 1.01, Ala 1.07, Val 1.0,
Leu 1.0, 23.68 838.5 Arg 1.90 10-40% (40 min.) 27
c(MePhe-Leu-Asp-Val-D-Ala-D-Arg-D-Arg) Asp 1.05, Ala 1.0, Val 0.97,
Leu 0.98, 26.48 871.5 Arg 2.0 10-40% (40 min.) 28
c(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Ala) Asp 1.03, Ala 0.99, Val 1.0,
Leu 1.0, 23.16 838.4 Arg 1.97. 10-40% (30 min.) 29
c(MePhe-Leu-Asp-Val-D-Arg-D-Arg-D-Ala) Asp 1.02, Ala 1.0, Val 0.98,
Leu 0.99, 27.38 872.5 Arg 1.96. 10-40% (30 min.) 30
c(MeIle-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg) Asp 1.03, Ala 1.98,
Val 0.97, Leu 1.0, 22.55 909.6 Arg 2.05. 10-40% (30 min.) 31
c(MePhe-Leu-Asp-Val-D-Ala-D-Ala-D-Arg-D-Arg) Asp 1.06, Ala 2.09,
Val 1.0, Leu 1.0, 24.35 943.5 Arg 1.99 10-40% (30 min.) 32
c(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Ala-D-Ala) Asp 1.0, Ala 2.08, Val
0.96, Leu 1.05, 24.33 909.5 Arg 1.95. 10-40% (30 min.) 33
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.5 CO) Asp 1.01, Val
0.98, Leu 0.99, Ahx 23.16 724.3 0.99, Arg 1.02 10-40% (30 min.) 34
c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.2 --S--(CH.sub.2).sub.2
-- Asp 1.03, Val 0.96, Leu 0.99, Arg 1.02 23.23 742 CO) 10-40% (30
min.) 35 c(D-Arg-MeIle-Leu-Asp-Val-NH(CH.sub.2).sub.2 --S--CH.sub.2
--CO) Asp 0.97, Val 0.97, Leu 1.01, Arg 1.05 23.23 728.4 10-40% (30
min.) 36 c(MeIle-Leu-Asp-Val-D-Arg-NH(CH.sub.2).sub.2 --S--CH.sub.2
CO) Asp 1.0, Val 1.02, Leu 1.04, Arg 1.01. 17.37 728.4 20-40% (40
min.) 37 c(MeIle-Leu-Asp-Val-NH--CH.sub.2 --CH.sub.2 --S--CH.sub.2
--CO-D- Asp 1.0, Val 1.0, Leu 1.01, Arg 2.0. 13.0 884.4 Arg-D-Arg)
20-40% (40 min.) 38 c(MePhe-Leu-Asp-Val-NH--CH.sub.2 --CH.sub.2
--S--CH.sub.2 --CO-D- Asp 1.0, Val 0.95, Leu 0.98, Arg 1.97. 16.0
918.4 Arg-D-Arg) 20-40% (40 min.) Analytical HPLC was carried out
using either a reverse phase (C.sub.18) Vydac column (218TP54, 4.6
.times. 250 mm) or a Novapak column (3.9 .times. 150 mm). Unless
otherwise stated in the above table a Vydac column was used for the
compound. The solvent system consisted of water and acetonitrile
(each containing 0.1% trifluoroacetic acid). The column was eluted
using a gradient (solvent ratio and time shown in the table) with
increasing concentrations of acetonitrile run at a rate of #1
ml/minute. The presence of some of the unnatural amino acids was
observed in the amino acid analysis but the quantities were not
estimated.
* * * * *